Methods for quantitative amplification and detection over a wide dynamic range

ABSTRACT

Disclosed are compositions and methods for making differentiable amplicon species at unequal ratios using a single amplification system in a single vessel. The number of differentiable amplicons and their ratios to one another are chosen to span the required linear dynamic range for the amplification reaction and to accommodate limitations of the measuring system used to determine the amount of amplicon generated. Unequal amounts of distinguishable amplicon species are generated by providing unequal amounts of one or more amplification reaction components (e.g., distinguishable amplification oligomers, natural and unnatural NTP in an NTP mix, or the like). The amount of target nucleic acid present in a test sample is determined using the linear detection range generated from detection of one or more amplicon species having an amount within the dynamic range of detection.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/562,922, filed Dec. 8, 2014, now U.S. Pat. No. 9,347,098, which is acontinuation of Ser. No. 14/064,427, filed Oct. 28, 2013, now U.S. Pat.No. 8,932,817, which is a continuation of U.S. application Ser. No.12/840,971, filed Jul. 21, 2010, now U.S. Pat. No. 8,628,924, whichclaims the benefit of U.S. Provisional Application Nos. 61/227,339,filed Jul. 21, 2009, and 61/230,938, filed Aug. 3, 2009 each of which ishereby incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety.

FIELD

The present application relates to compositions and methods foramplifying and detecting a nucleic acid sequence that may be present ina test sample, more particularly, for amplifying and quantitativelydetecting a nucleic acid sequence over a wide dynamic range for whichthe target nucleic acid sequence may be present in the sample.

BACKGROUND

Methods that allow accurate detection and quantitation of nucleic acidsequences are invaluable tools for diagnosis and treatment of a widerange of diseases. For example, detection and quantitation of aninfectious agent's nucleic acid sequence can provide diagnostic orprognostic information or allow a physician to monitor a patient'sresponse to therapy. Further, accurate detection of low levels ofviremia in infections such as human immunodeficiency virus (HIV) orhepatitis C virus helps to prevent spread of the virus, to estimateprognosis and response to therapy, and to detect the emergence ofdrug-resistant viruses in treated individuals. It is equally importantto accurately measure high levels of viremia in untreated patients toestablish initial infection levels.

Methods for amplifying a target nucleic acid sequence that may bepresent in a test sample are known and include methods such as thepolymerase chain reaction (PCR; e.g., U.S. Pat. Nos. 4,683,195,4,683,202, 4,965,188, 6,040,166, 6,197,563 and 6,514,736); reversetranscription polymerase chain reaction (RT-PCR; e.g., U.S. Pat. Nos.5,310,652 and 5,322,770); transcription-mediated amplification (TMA;e.g., U.S. Pat. Nos. 5,399,491, 5,824,518 and 7,374,885); ligase chainreaction (LCR; e.g., U.S. Pat. Nos. 5,427,930 and 5,516,663); stranddisplacement amplification (SDA; e.g., U.S. Pat. Nos. 5,422,252,5,547,861 and 5,648,211); rolling circle amplification (RCA; e.g., U.S.Pat. No. 5,648,245 and U.S. Pat. No. 5,854,033); helicase-dependentamplification (HDA; e.g., U.S. Pat. Nos. 7,282,328 and 7,662,594); andnucleic acid sequence based amplification (NASBA; e.g., U.S. Pat. No.5,130,238).

Methods for detecting non-amplified or amplified target nucleic acidsequences that may be present in a test sample are also known. Somedetection methods are “homogeneous” methods that do not requireseparation of a detection agent associated with the target nucleic acidfrom the detection agent that is not associated with the target nucleicacid. Such methods include use of intercalating dyes (e.g., U.S. Pat.Nos. 5,312,921, 5,814,447, 6,063,572, 6,541,205 and 6,569,627), aHybridization Protection Assay (HPA; e.g., U.S. Pat. No. 5,283,174), anduse of molecular beacon probes (e.g., U.S. Pat. No. 5,925,517) ormolecular torch probes (e.g., U.S. Pat. No. 6,361,945). Other methodsare “heterogeneous” and require physical separation of the detectionagent associated with the target nucleic acid from the detection agentnot associated with the target nucleic acid. One of the most commonheterogeneous methods involves the capture of target nucleic acids ontoa solid support, hybridization of a labeled detection probe, and washingunder appropriately stringent conditions to remove non-specificallybound probe. The label (e.g, a radioisotope) that remains bound to thesupport is then measured.

While currently available amplification techniques may providesufficient sensitivity for some applications, other diagnostic andtherapeutic situations require more sensitivity than is available fromthese available methods. Furthermore, under certain circumstances, it isdesirable to determine whether the nucleic acid sequence is present at ahigh or low concentration level. Thus, there is a need for methodsthrough which target nucleic acids in a test sample can be detected andquantified accurately over a relatively large dynamic range in a singleexperiment.

Nucleic acid analytes of interest from a specimen may be present atconcentrations that are less than can be detected by routine methods.This problem is often circumvented by implementing one of severalnucleic acid amplification techniques (vide supra) prior to detection.The nucleic acid analytes of interest from a specimen may also bepresent in very small numbers (e.g., 1, 7, 29, etc., or zero in the caseof a non-infected specimen) or larger numbers ranging into the millions,billions or more. Therefore, it is critical for quantification that thenucleic acid analytes are amplified proportionally to accurately reflecttheir initial numbers. Attempts to achieve reproducible quantitation ofnucleic acids over a dynamic range by using nucleic acid amplificationmethods has been a challenging endeavor and a number of problems havebeen encountered including, for example, the need for amplificationinternal standards and (even very slightly) different amplificationefficiencies of internal standards (Clin. Chem. 40(4): 630-636 (1994);Clin. Chem. 41(8): 1065-1067 (1995); Biochim. Biophys. Acta 1219(2):493-498 (1994); Biotechniques 15(1): 134-139 (1993); J. Infect. Dis.165(6): 1119-1123 (1992); Proc. Nat. Acad. Sci. USA 86(24): 9717-9721(1989)). Two basic approaches have been used to solve these problems,with mixed success.

In the first basic approach, the concentration of a target nucleic acidsequence present in a test sample is determined based on the rate atwhich amplicons are produced over time. An example of this approach isknown as “real-time PCR;” increasing amplicon levels are measured withprobes that change their fluorescent properties in response toincreasing amplicon concentration. The time it takes for the amplicon toreach a predefined level is correlated with the concentration of thetarget nucleic acid sequence present in the sample by comparing theresults of the experimentally detected amplicon to results fromstandards that contain a range of known amounts of the target nucleicacid sequences. Multiple standards may be used, often ranging from thelow to high ends of expected responses from the analyte of unknownconcentration. In amplification systems in which the reaction is dividedinto cycles, such as the PCR or LCR, precision is limited by the amountof amplification that occurs within each cycle; for the PCR, the amountis theoretically a two-fold increase per cycle. However, foramplification systems that lack discrete amplification cycles, such asTMA or SDA, rate changes may be difficult to measure accurately becauseamplicon concentration can change rapidly over a very short period oftime. The accuracy of time and amplicon concentration measurementsgreatly affects the ability to correlate measured results with aninitial target nucleic acid concentration with any degree of precision.

In the second basic approach, the amplification reaction is run for afixed time, and the amount of amplicon produced is measured andcorrelated to the concentration of target nucleic acid sequence presentin the test sample. Such methods are sometimes referred to as “endpoint”methods because the amplicons are measured at a single point at the endof the reaction rather than at multiple time points during theamplification reaction. These methods typically require the use of anexcess of nucleic acid probe relative to the target nucleic acid so thatthe amount of probe hybridized relates directly to the amount of targetpresent and avoids signal saturation due to excess target, referred toas “target saturation” (e.g., U.S. Pat. No. 4,851,330). If targetsaturation occurs, the probe signal reaches a plateau and the upper endof the dynamic range is truncated. When large amounts of amplicon areproduced, even larger amounts of probe must be used to extend thedynamic range, and the amplicon-probe hybridization signal may exceedthe detector's capacity for accurate detection. Furthermore, thebackground signal is often elevated as probe concentration increases,which limits sensitivity of detection at the lower end of the dynamicrange. Lowering the specific activity of the probe can be used to reducethe background signal; however, the sensitivity of detection at lowtarget levels is also often decreased. Thus, optimization of endpointdetection may reduce accuracy on one or both ends of the dynamic range.

Generally, while known amplification systems have been optimized toproduce amplicons quantitatively over a large dynamic range, detectionsystems have been unable to perform accurately over that same broadrange. This limitation requires additional steps in the process, such asserial dilution of the amplicons or physical separation steps, in orderto reduce non-specific background signal and to measure the ampliconconcentration across the full dynamic range. Physical separation ordilution steps increase the complexity and length of the test procedure.Manipulating amplicon products increases the danger ofcross-contaminating samples or amplification reaction mixtures, whichcould lead to false positive results. In addition, amplicon manipulationincreases the possibility of errors in quantification, including falsenegative results. Ideally, a nucleic acid detection and quantitationsystem should include an amplification reaction that generatesmeasurable amounts of one or more amplicons reproducibly andproportionately across a wide dynamic range, as well as a detectionsystem that can quantitate the amplicons across the same dynamic range.

In certain known methods, a nucleic acid hybridization reaction is usedto determine the amount of amplicon by correlating either the rate ofhybridization or the extent of hybridization to the amount of ampliconpresent. However, kinetic measurements of hybridization pose many of thesame disadvantages of kinetic measurements of amplification reactionsand are thus not preferred.

In some variations of known in vitro nucleic acid amplificationprocedures, researchers have sought to achieve an extended dynamic rangeby individually adjusting the efficiencies of amplification of one ormore target sequences by optimizing the reaction conditions for eachtarget nucleic acid. However, such optimization generally requirescomplex and extensive experimentation and may not yield reproducibleresults because of slight differences between samples, presence ofinhibitors in reaction mixtures, variations in test reagents and/ortheir quantities in individual reaction mixtures, or conditions in whichthe reactions are performed. Thus, there remains a need to extend thedynamic range of quantitative endpoint assays that does not requireexcess experimentation and provides a robust system with wide dynamicrange.

Compositions and methods that respond to this need would allowquantitative measurement of a desired target nucleic acid in a sample byusing nucleic acid amplification and detection of the amplified productsover an extended dynamic range. The compositions and methods describedherein provide a simple solution to problems associated with previousquantitative nucleic acid amplification and detection methods.

SUMMARY

The present application provides compositions and methods for makingmultiple differentiable amplicon species at unequal ratios using asingle amplification system in a single vessel. The number ofdifferentiable amplicon species and their ratios to one another arechosen to span the required linear dynamic range for the amplificationreaction and to accommodate limitations of the measuring system used todetermine the amount of any one amplicon generated. Unequal amounts ofdistinguishable amplicon species are generated by providing unequalamounts of one or more amplification reaction components (e.g., unequalamounts of distinguishable amplification oligomers, unequal amounts ofnatural and unnatural NTP in an NTP mix, or the like). If small amountsof target nucleic acid are present in the sample, then the more abundantamplicons species is detectable within the linear dynamic range, whilethe lesser abundant amplicon species is/are below the detectionthreshold. If greater amounts of target nucleic acid are present in thesample, then less abundant amplicons species become detectable withinthe linear dynamic range. Here, detection of target amounts can bedetermined using the detection ranges generated from two or moreamplicon species. At higher amounts of target nucleic acid present inthe sample, the more abundant amplicon species can saturate thedetection system; however, lesser abundance amplicon species are stilldetectable within their linear dynamic ranges. Therefore, for twoamplicons, when a large amount of target nucleic acid is present in atest sample, large amounts of amplicon 1 are generated but smalleramounts of amplicon 2 is also made. If the amount of amplicon 1 isoutside the dynamic range of the detection system, then the amounts ofamplicon 2 may lie within the dynamic range and can be measured. Theamount of original target nucleic acid sequence may then be calculatedfrom the amount of amplicon 2. Additional amplicons can be made tofurther extend the dynamic range (amplicon 3, etc.).

The present application provides a method for detecting a target nucleicacid in a sample comprising the steps of: providing a sample suspectedof containing a target nucleic acid; generating from the target nucleicacid, a defined ratio of at least two differentiable amplicon species,wherein the generating step is performed in a single vessel; anddetecting the presence and amount of each generated amplicon species,wherein a first amplicon species is detectable in a first linear rangerepresenting a first concentration of target nucleic acid in the sampleto a second concentration of target nucleic acid in the sample and asecond amplicon species is detectable in a second linear rangerepresenting a third concentration of target nucleic acid in the sampleto a fourth concentration of target nucleic acid in the sample, andwherein, the first concentration is less that the third concentration,which is less than the second concentration, which is less than thefourth concentration such that said first and second linear rangesoverlap and provide an extended dynamic range for determining thepresence and amount of the target nucleic acid in the sample, andwherein the detecting step is performed in a single vessel. Preferably,the single well used in the amplifying step and the single vessel usedin the vessel are the same vessel. Thus, the amplification and detectionreactions take place in the same vessel.

In some aspects of the method, a third differentiable amplicon speciesis generated. The third differentiable amplicon species will provide athird linear range that is different than the first and the secondlinear ranges, thereby further extending the dynamic range fordetermining the presence and amount of the target nucleic acid in thesample. In some aspects, the third amplicon is detectable in a linearrange representing a representing a fifth concentration of targetnucleic acid in the sample to a sixth concentration of target nucleicacid in the sample, wherein the first concentration is less than thethird concentration, which is less than the second concentration, whichis less than the fifth concentration, which is less than the fourthconcentration, which is less than the sixth concentrations such that thefirst, second and third linear ranges overlap and provide an extendeddynamic range for determining the presence and amount of said targetnucleic acid in said sample. In some aspects n differentiable ampliconspecies are generated to provide n overlapping linear ranges to providean extended dynamic range; n being equal to a positive whole number.

In some aspects of the methods the at least two differentiable ampliconspecies are generated using a single amplification oligomer thathybridizes to one strand of the target nucleic acid and at least twoamplification oligomers that hybridize to the complementary strand ofthe target nucleic acid, wherein the at least two amplificationoligomers hybridizing to a complementary strand of the target nucleicacid are provided in unequal amounts. In some aspects, the singleamplification oligomer is a promoter-based amplification oligomer. Insome aspects, the single amplification oligomer is a promoter-provider.In some aspects, the single amplification oligomer is a promoter-primer.In some aspects, the single amplification oligomer is a primer. In someaspects, the at least two amplification oligomers that hybridize to thecomplementary strand of the target nucleic acid are promoter-basedamplification oligomers. In some aspects, the at least two amplificationoligomers that hybridize to the complementary strand of the targetnucleic acid are promoter-primers. In some aspects, the at least twoamplification oligomers that hybridize to the complementary strand ofthe target nucleic acid are promoter-providers. In some aspects, the atleast two amplification oligomers that hybridize to the complementarystrand of the target nucleic acid are primers. In some aspects, the atleast two differentiable amplicon species are generated using a singlepromoter based amplification oligomer that hybridizes to one strand ofthe target nucleic acid and at least two primer that hybridize to thecomplementary strand of the target nucleic acid. The singlepromoter-based amplification oligomer is a promoter-provider in someaspects of the methods. The single promoter-based amplificationoligomer—is a promoter primer in some aspects of the methods. In someaspects, the at least two differentiable amplicon species are generatedusing a single primer that hybridizes to one strand of the targetnucleic acid and at least two promoter-based amplification oligomersthat hybridize to the complementary strand of the target nucleic acid.The at least two promoter-based amplification oligomers arepromoter-providers in some aspects of the methods. The at least twopromoter-based amplification oligomers are promoter primers in someaspects of the methods. In some aspects, the at least two differentiableamplicon species are generated using a single primer that hybridizes toone strand of the target nucleic acid and at least two primers thathybridize to the complementary strand of the target nucleic acid.

In some aspects of the methods wherein the at least two differentiableamplicon species are generated using a single amplification oligomerthat hybridizes to one strand of the target nucleic acid and unequalamounts of at least two amplification oligomers that hybridize to thecomplementary strand of the target nucleic acid, the at least twoamplification oligomers hybridizing to the complementary strand of thetarget nucleic acid hybridize to distinct sequences of said targetnucleic acid, thereby generating from the target nucleic acid, a definedratio of at least two differentiable amplicon species that differ inlength. In some aspects, the distinct sequences of the target nucleicacid to which the at least two amplification oligomers hybridize, arenon-overlapping sequences. In some aspects, the distinct sequences ofthe target nucleic acid to which the at least two amplificationoligomers hybridize, are overlapping sequences.

In some aspects of the methods wherein the at least two differentiableamplicon species are generated using a single amplification oligomerthat hybridizes to one strand of the target nucleic acid and unequalamounts of at least two amplification oligomers that hybridize to thecomplementary strand of the target nucleic acid, the at least twoamplification oligomers hybridizing to the complementary strand of thetarget nucleic acid have distinct nucleic acid sequences, therebygenerating from the target nucleic acid, a defined ratio of at least twodifferentiable amplicon species that differ in nucleic acid composition.In some aspects, the at least two amplification oligomers aresubstantially identical in nucleotide sequence except that oneamplification oligomer has one or more of: at least one nucleotidemismatch, at least one nucleotide insertion, at least one nucleotidedeletion and combinations thereof, thereby generating from said targetnucleic acid, a defined ratio of at least two differentiable ampliconspecies that differ in nucleic acid composition. In some aspects, the atleast two amplification oligomers each contain at least one insertion,wherein the at least one insertion in one of the at least twoamplification oligomers is different from the at least one insertion ofthe other at least one amplification oligomers. In some aspects, each ofthe at least one amplification oligomers comprises a unique unhybridizednucleotide sequence that is at least one nucleobase in length and thatis joined to the 5′ end of the target hybridizing region of theamplification oligomer.

In some aspects of the methods, the two differentiable amplicon speciesare generated using an amplification method wherein said amplificationmethod generates RNA amplicon species and DNA amplicon species toprovide at least two differentiable amplicons that differ in ribosesugar composition. In some aspects, the amplification method is anisothermal amplification method that uses at least one promoter-basedamplification oligomer to generate a greater amount of RNA ampliconspecies that of DNA amplicon species.

In some aspects of the methods, the two differentiable amplicon speciesare generated using a dNTP mixture wherein at least one of said dNTPspecies in said mix is provided in both a native form and in an analogform with one form in excess of the other form to provide at least twodifferentiable amplicons that differ in nucleic acid composition. Insome aspects, the dNTP mix contains native dATP and an analog of dATP.In some aspects, the dNTP mix contains native dCTP and an analog ofdCTP. In some aspects, the dNTP mix contains native dTTP and an analogof dTTP. In some aspects, the dNTP mix contains native dGTP and ananalog of dGTP. In some aspects, the dNTP mix contains native dUTP andan analog of dUTP.

In some aspects, the amplification step is performed with an isothermalamplification reaction. In some aspects, the amplification step isperformed with an cyclical amplification reaction. In some aspects, theamplification step is performed with a TMA amplification reaction. Insome aspects, the amplification step is performed with a NASBAamplification reaction. In some aspects, the amplification step isperformed with a PCR amplification reaction. In some aspects, theamplification step is performed with a Reverse Transcription (RT)-PCRamplification reaction. In some aspects, the amplification step isperformed with a real time amplification reaction. In some aspects, theamplification step is performed with a rolling circle amplificationreaction. In some aspects, the amplification step is performed with ahelicase dependent amplification reaction.

The present application provides a method for amplifying a targetnucleic acid sequence in a test sample comprising: (a) contacting thetest sample with a nucleic acid amplification reaction mixturecomprising at least two primers, wherein the at least two primershybridize to the same strand of the target nucleic acid sequence buthybridize to distinct nucleotide sequences on the strand, and each ofthe at least two primers is present in the reaction mixture in adifferent amount; and (b) subjecting the reaction mixture toamplification conditions under which each of the at least two primerssimultaneously and independently produces one of each of twodistinguishable amplicons from the target nucleic acid sequence, whereinthe number of copies of each amplicon produced depends on the amount ofeach of the at least two primers in the reaction mixture, and the numberof copies of each amplicon produced differs by at least two orders ofmagnitude.

In some aspects, each of the at least two primers further differ fromone another by having one or more of (i) one of the amplificationoligomers has a modified target hybridizing sequence and the other doesnot and/or (ii) each of the amplification oligomers has a differentunique unpaired 5′ sequence.

The present application provides a method for quantitating a targetnucleic acid sequence in a test sample comprising: (a) contacting thetest sample with a nucleic acid amplification reaction mixturecomprising at least two primers, wherein the at least two primershybridize to the same strand of the target nucleic acid sequence buthybridize to distinct nucleotide sequences on the strand, and each ofthe at least two primers is present in the reaction mixture in adifferent amount; (b) subjecting the reaction mixture to amplificationconditions under which each of the at least two primers simultaneouslyand independently produces one of at least two amplicons, wherein thenumber of copies of each amplicon produced differs by at least twoorders of magnitude; (c) hybridizing the at least two amplicons with atleast two probes, wherein each of the at least two probes is specific toone of the at least two amplicons, each probe is detectable within adetection range, wherein the sum of the detection ranges for each of theat least two probes is greater than the detection range for each probe,such that the at least two probes together are detectable across a widedynamic range, and the at least two probes produce distinguishabledetection signals; (d) detecting at least one of the at least twoprobes; and (e) determining the initial amount of target nucleic acidsequence in the test sample.

In some aspects, each of the at least two primers further differ fromone another by having one or more of (i) one of the amplificationoligomers has a modified target hybridizing sequence and the other doesnot and/or (ii) each of the amplification oligomers has a differentunique unpaired 5′ sequence.

The present application further provides a method for quantitating anucleic acid in a test sample comprising: (a) providing a test samplesuspected of containing a target nucleic acid in an amount T₁; (b)contacting the test sample with a nucleic acid amplification reactionmixture comprising at least two primers, wherein the at least twoprimers hybridize to the same strand of the target nucleic acid sequencebut each primer hybridizes to a distinct nucleotide sequence on thestrand, and wherein each primer is present in the reaction mixture in adifferent amount. In one aspect, each P_(x) differs by at least twoorders of magnitude, and where x is an integer between 1 and the numberof primers in the mixture. (c) Subjecting the reaction mixture toamplification conditions under which each of the at least two primerssimultaneously and independently produces one of at least two amplicons,wherein for each amplicon, a number of copies A_(x) is produced, andwherein each A_(x) differs by an amount that is approximately the sameas the amount of difference in each P_(x) for the primers present in themix. In one aspect, the A_(x) for each amplicon differs by at least twoorders of magnitude. (d) Hybridizing the at least two amplicons with atleast two probes, wherein each of the at least two probes is specific toone of the at least two amplicons, each probe is detectable within alinear detection range C_(x(a)) to C_(x(b)), wherein C_(x(a)) is theminimum detectable number of copies of amplicon and C_(x(b)) is themaximum detectable number of copies of amplicon for probe x, wherein foreach probe, C_(x+1(a)) is greater than C_(x(a)) and C_(x+1(b)) isgreater than C_(x(b)) such that the at least two probes together aredetectable across a wide dynamic range, wherein for each amplicon-probecombination, A_(x) is between C_(x(a)) and C_(x(b)), and wherein the atleast two probes produce distinguishable detection signals; (e)detecting at least one of the at least two probes; and (f) determiningthe initial amount of target nucleic acid sequence in the test sample.

In some aspects, each of the at least two primers further differ fromone another by having one or more of (i) one of the amplificationoligomers has a modified target hybridizing sequence and the other doesnot and/or (ii) each of the amplification oligomers has a differentunique unpaired 5′ sequence.

The present application further provides a composition for amplifying atarget nucleic acid sequence in a test sample comprising at least twoprimers, wherein (a) the at least two primers hybridize to the samestrand of the target nucleic acid sequence but hybridize to distinctnucleotide sequences on the strand, and each of the at least two primersis present in the reaction mixture in a different amount; and (b) underamplification conditions, each of the at least two primerssimultaneously and independently produces one of each of two detectableamplicons from the target nucleic acid sequence, wherein the number ofcopies of each amplicon produced depends on the amount of each of the atleast two primers in the reaction mixture, the number of copies of eachamplicon produced differs by at least two orders of magnitude, and theamplicons are detectable across a wide dynamic range.

In some aspects, each of the at least two primers further differ fromone another by having one or more of (i) one of the amplificationoligomers has a modified target hybridizing sequence and the other doesnot and/or (ii) each of the amplification oligomers has a differentunique unpaired 5′ sequence.

The present application further provides a nucleic acid amplificationreaction mixture comprising at least two primers, wherein: (a) the atleast two primers hybridize to the same strand of a target nucleic acidsequence but each primer hybridizes to a distinct nucleotide sequence onthe strand; wherein each primer is present in the reaction mixture at adifferent amount P_(x), where x is an integer between 1 and the numberof primers in the mixture; (b) under amplification conditions, eachprimer simultaneously and independently produces one of at least twoamplicons from the target nucleic acid sequence, wherein for eachamplicon, a number of copies A_(x) is produced, and wherein each A_(x)differs by an amount that is approximately same as the amount ofdifference in each P_(x) of the at least two primers. In one aspect, thedifference in the A_(x) for each amplicon is at least two orders ofmagnitude. (c) The at least two amplicons are detectable byhybridization with at least two probes, wherein each of the at least twoprobes is specific to one of the at least two amplicons, and each probeis detectable within a linear detection range C_(x(a)) to C_(x(b)),wherein C_(x(a)) is the minimum detectable number of copies of ampliconand C_(x(b)) is the maximum detectable number of copies of amplicon forprobe x, wherein for each probe, C_(x+1(a)) is greater than C_(x(a)) andC_(x+1(b)) is greater than C_(x(b)) such that the at least two probestogether are detectable across a wide dynamic range, wherein for eachamplicon-probe combination, A_(x) is between C_(x(a)) and C_(x(b)), andwherein the at least two probes produce distinguishable detectionsignals.

In some aspects, each of the at least two primers further differ fromone another by having one or more of (i) one of the amplificationoligomers has a modified target hybridizing sequence and the other doesnot and/or (ii) each of the amplification oligomers has a differentunique unpaired 5′ sequence.

The present application provides a method for amplifying a targetnucleic acid sequence in a test sample comprising: (a) contacting thetest sample with a nucleic acid amplification reaction mixturecomprising at least two amplification oligomers, wherein the at leasttwo amplification oligomers hybridize to the same strand of the targetnucleic acid sequence but hybridize to distinct nucleotide sequences onthe strand, and each of the at least two amplification oligomers ispresent in the reaction mixture in a different amount; and (b)subjecting the reaction mixture to amplification conditions under whicheach of the at least two amplification oligomers simultaneously andindependently produces one of each of two distinguishable amplicons fromthe target nucleic acid sequence, wherein the number of copies of eachamplicon produced depends on the amount of each of the at least twoamplification oligomers in the reaction mixture, and the number ofcopies of each amplicon produced differs by at least two orders ofmagnitude.

In some aspects, the at least two amplification oligomers arepromoter-based amplification oligomers. In some aspects, each of the atleast two amplification oligomers further differ from one another byhaving one or more of (i) one of the amplification oligomers has amodified target hybridizing sequence and the other does not and/or (ii)each of the amplification oligomers has a different unique unpaired 5′sequence.

The present application provides a method for quantitating a targetnucleic acid sequence in a test sample comprising: (a) contacting thetest sample with a nucleic acid amplification reaction mixturecomprising at least two amplification oligomers, wherein the at leasttwo amplification oligomers hybridize to the same strand of the targetnucleic acid sequence but hybridize to distinct nucleotide sequences onthe strand, and each of the at least two amplification oligomers ispresent in the reaction mixture in a different amount; (b) subjectingthe reaction mixture to amplification conditions under which each of theat least two amplification oligomers simultaneously and independentlyproduces one of at least two amplicons, wherein the number of copies ofeach amplicon produced differs by at least two orders of magnitude; (c)hybridizing the at least two amplicons with at least two probes, whereineach of the at least two probes is specific to one of the at least twoamplicons, each probe is detectable within a detection range, whereinthe sum of the detection ranges for each of the at least two probes isgreater than the detection range for each probe, such that the at leasttwo probes together are detectable across a wide dynamic range, and theat least two probes produce distinguishable detection signals; (d)detecting at least one of the at least two probes; and (e) determiningthe initial amount of target nucleic acid sequence in the test sample.

In one aspect, the at least two amplification oligomers arepromoter-based amplification oligomers. In some aspects, each of the atleast two amplification oligomers further differ from one another byhaving one or more of (i) one of the amplification oligomers has amodified target hybridizing sequence and the other does not and/or (ii)each of the amplification oligomers has a different unique unpaired 5′sequence.

The present application further provides a method for quantitating anucleic acid in a test sample comprising: (a) providing a test samplesuspected of containing a target nucleic acid in an amount T₁; (b)contacting the test sample with a nucleic acid amplification reactionmixture comprising at least two amplification oligomers, wherein the atleast two amplification oligomers hybridize to the same strand of thetarget nucleic acid sequence but each amplification oligomers hybridizesto a distinct nucleotide sequence on the strand, and wherein eachamplification oligomers is present in the reaction mixture in adifferent amount. In one aspect, each P_(x) differs by at least twoorders of magnitude, and where x is an integer between 1 and the numberof amplification oligomers in the mixture. (c) Subjecting the reactionmixture to amplification conditions under which each of the at least twoamplification oligomers simultaneously and independently produces one ofat least two amplicons, wherein for each amplicon, a number of copiesA_(x) is produced, and wherein each A_(x) differs by an amount that isapproximately the same as the amount of difference in each P_(x) for theat least two amplification oligomers hybridizing to the same strand ofthe target nucleic acid sequence present in the mix. In one aspect, theA_(x) for each amplicon differs by at least two orders of magnitude. (d)Hybridizing the at least two amplicons with at least two probes, whereineach of the at least two probes is specific to one of the at least twoamplicons, each probe is detectable within a linear detection rangeC_(x(a)) to C_(x(b)), wherein C_(x(a)) is the minimum detectable numberof copies of amplicon and C_(x(b)) is the maximum detectable number ofcopies of amplicon for probe x, wherein for each probe, C_(x+1(a)) isgreater than C_(x(a)) and C_(x+1(b)) is greater than C_(x(b)) such thatthe at least two probes together are detectable across a wide dynamicrange, wherein for each amplicon-probe combination, A_(x) is betweenC_(x(a)) and C_(x(b)), and wherein the at least two probes producedistinguishable detection signals; (e) detecting at least one of the atleast two probes; and (f) determining the initial amount of targetnucleic acid sequence in the test sample.

In some aspects, the at least two amplification oligomers arepromoter-based amplification oligomers. In some aspects, each of the atleast two amplification oligomers further differ from one another byhaving one or more of (i) one of the amplification oligomers has amodified target hybridizing sequence and the other does not and/or (ii)each of the amplification oligomers has a different unique unpaired 5′sequence.

The present application further provides a composition for amplifying atarget nucleic acid sequence in a test sample comprising at least twoamplification oligomers, wherein: (a) the at least two amplificationoligomers hybridize to the same strand of the target nucleic acidsequence but hybridize to distinct nucleotide sequences on the strand,and each of the at least two amplification oligomers is present in thereaction mixture in a different amount; and (b) under amplificationconditions, each of the at least two amplification oligomerssimultaneously and independently produces one of each of two detectableamplicons from the target nucleic acid sequence, wherein the number ofcopies of each amplicon produced depends on the amount of each of the atleast two amplification oligomers in the reaction mixture, the number ofcopies of each amplicon produced differs by at least two orders ofmagnitude, and the amplicons are detectable across a wide dynamic range.

In some aspects, the at least two amplification oligomers arepromoter-based amplification oligomers. In some aspects, each of the atleast two amplification oligomers further differ from one another byhaving one or more of (i) one of the amplification oligomers has amodified target hybridizing sequence and the other does not and/or (ii)each of the amplification oligomers has a different unique unpaired 5′sequence.

The present application further provides a nucleic acid amplificationreaction mixture comprising at least two amplification oligomers,wherein: (a) the at least two amplification oligomers hybridize to thesame strand of a target nucleic acid sequence but each amplificationoligomer hybridizes to a distinct nucleotide sequence on the strand;wherein each primer is present in the reaction mixture at a differentamount P_(x), where x is an integer between 1 and the number of primersin the mixture; (b) under amplification conditions, each primersimultaneously and independently produces one of at least two ampliconsfrom the target nucleic acid sequence, wherein for each amplicon, anumber of copies A_(x) is produced, and wherein each A_(x) differs by anamount that is approximately same as the amount of difference in eachP_(x) of the at least two amplification oligomers hybridizing to thesame strand of the target nucleic acid sequence. In one aspect, thedifference in the A_(x) for each amplicon is at least two orders ofmagnitude; (c) the at least two amplicons are detectable byhybridization with at least two probes, wherein each of the at least twoprobes is specific to one of the at least two amplicons, and each probeis detectable within a linear detection range C_(x(a)) to C_(x(b)),wherein C_(x(a)) is the minimum detectable number of copies of ampliconand C_(x(b)) is the maximum detectable number of copies of amplicon forprobe x, wherein for each probe, C_(x+1(a)) is greater than C_(x(a)) andC_(x+1(b)) is greater than C_(x(b)) such that the at least two probestogether are detectable across a wide dynamic range, wherein for eachamplicon-probe combination, A_(x) is between C_(x(a)) and C_(x(b)), andwherein the at least two probes produce distinguishable detectionsignals.

In some aspects, the at least two amplification oligomers arepromoter-based amplification oligomers. In some aspects, each of the atleast two amplification oligomers further differ from one another byhaving one or more of (i) one of the amplification oligomers has amodified target hybridizing sequence and the other does not and/or (ii)each of the amplification oligomers has a different unique unpaired 5′sequence.

The present application provides a method for amplifying a targetnucleic acid sequence in a test sample comprising: (a) contacting thetest sample with a nucleic acid amplification reaction mixturecomprising at least two amplification oligomers, wherein the at leasttwo amplification oligomers hybridize to the same sequence on the samestrand of the target nucleic acid sequence but each of the at least twoamplification oligomers differ from one another by having one or more of(i) one of the amplification oligomers has a modified target hybridizingsequence and the other does not and/or (ii) each of the amplificationoligomers has a different unique unpaired 5′ sequence, and each of theat least two amplification oligomers is present in the reaction mixturein a different amount; and (b) subjecting the reaction mixture toamplification conditions under which each of the at least twoamplification oligomers simultaneously and independently produces one ofeach of two distinguishable amplicons from the target nucleic acidsequence, wherein the number of copies of each amplicon produced dependson the amount of each of the at least two amplification oligomers in thereaction mixture, and the number of copies of each amplicon produceddiffers by at least two orders of magnitude.

In some aspects, the at least two amplification oligomers arepromoter-based amplification oligomers. In some aspects, the at leasttwo amplification oligomers are primers.

The present application provides a method for quantitating a targetnucleic acid sequence in a test sample comprising: (a) contacting thetest sample with a nucleic acid amplification reaction mixturecomprising at least two amplification oligomers, wherein the at leasttwo amplification oligomers hybridize to the same sequence on the samestrand of the target nucleic acid sequence but each of the at least twoamplification oligomers differ from one another by having one or more of(i) one of the amplification oligomers has a modified target hybridizingsequence and the other does not and/or (ii) each of the amplificationoligomers has a different unique unpaired 5′ sequence, and each of theat least two amplification oligomers is present in the reaction mixturein a different amount; (b) subjecting the reaction mixture toamplification conditions under which each of the at least twoamplification oligomers simultaneously and independently produces one ofat least two amplicons, wherein the number of copies of each ampliconproduced differs by at least two orders of magnitude; (c) hybridizingthe at least two amplicons with at least two probes, wherein each of theat least two probes is specific to one of the at least two amplicons,each probe is detectable within a detection range, wherein the sum ofthe detection ranges for each of the at least two probes is greater thanthe detection range for each probe, such that the at least two probestogether are detectable across a wide dynamic range, and the at leasttwo probes produce distinguishable detection signals; (d) detecting atleast one of the at least two probes; and (e) determining the initialamount of target nucleic acid sequence in the test sample.

In one aspect, the at least two amplification oligomers arepromoter-based amplification oligomers. In some aspects, the at leasttwo amplification oligomers are primers.

The present application further provides a method for quantitating anucleic acid in a test sample comprising: (a) providing a test samplesuspected of containing a target nucleic acid in an amount T₁; (b)contacting the test sample with a nucleic acid amplification reactionmixture comprising at least two amplification oligomers, wherein the atleast two amplification oligomers hybridize to the same sequence on thesame strand of the target nucleic acid sequence but each of the at leasttwo amplification oligomers differ from one another by having one ormore of (i) one of the amplification oligomers has a modified targethybridizing sequence and the other does not and/or (ii) each of theamplification oligomers has a different unique unpaired 5′ sequence, andwherein each amplification oligomers is present in the reaction mixturein a different amount. In one aspect, each P_(x) differs by at least twoorders of magnitude, and where x is an integer between 1 and the numberof amplification oligomers in the mixture. (c) Subjecting the reactionmixture to amplification conditions under which each of the at least twoamplification oligomers simultaneously and independently produces one ofat least two amplicons, wherein for each amplicon, a number of copiesA_(x) is produced, and wherein each A_(x) differs by an amount that isapproximately the same as the amount of difference in each P_(x) for theat least two amplification oligomers hybridizing to the same strand ofthe target nucleic acid sequence present in the mix. In one aspect, theA_(x) for each amplicon differs by at least two orders of magnitude. (d)Hybridizing the at least two amplicons with at least two probes, whereineach of the at least two probes is specific to one of the at least twoamplicons, each probe is detectable within a linear detection rangeC_(x(a)) to C_(x(b)), wherein C_(x(a)) is the minimum detectable numberof copies of amplicon and C_(x(b)) is the maximum detectable number ofcopies of amplicon for probe x, wherein for each probe, C_(x+1(a)) isgreater than C_(x(a)) and C_(x+1(b)) is greater than C_(x(b)) such thatthe at least two probes together are detectable across a wide dynamicrange, wherein for each amplicon-probe combination, A_(x) is betweenC_(x(a)) and C_(x(b)), and wherein the at least two probes producedistinguishable detection signals; (e) detecting at least one of the atleast two probes; and (f) determining the initial amount of targetnucleic acid sequence in the test sample.

In some aspects, the at least two amplification oligomers arepromoter-based amplification oligomers. In some aspects, the at leasttwo amplification oligomers are primers.

The present application further provides a composition for amplifying atarget nucleic acid sequence in a test sample comprising at least twoamplification oligomers, wherein: (a) the at least two amplificationoligomers hybridize to the same sequence of the same strand of thetarget nucleic acid sequence but each of the at least two amplificationoligomers differ from one another by having one or more of (i) one ofthe amplification oligomers has a modified target hybridizing sequenceand the other does not and/or (ii) each of the amplification oligomershas a different unique unpaired 5′ sequence, and each of the at leasttwo amplification oligomers is present in the reaction mixture in adifferent amount; and (b) under amplification conditions, each of the atleast two amplification oligomers simultaneously and independentlyproduces one of each of two detectable amplicons from the target nucleicacid sequence, wherein the number of copies of each amplicon produceddepends on the amount of each of the at least two amplificationoligomers in the reaction mixture, the number of copies of each ampliconproduced differs by at least two orders of magnitude, and the ampliconsare detectable across a wide dynamic range.

In some aspect, the at least two amplification oligomers arepromoter-based amplification oligomers. In some aspects, the at leasttwo amplification oligomers are primers.

The present application further provides a nucleic acid amplificationreaction mixture comprising at least two amplification oligomers,wherein: (a) the at least two amplification oligomers hybridize to thesame sequence of the same strand of a target nucleic acid sequence buteach of the at least two amplification oligomers differ from one anotherby having one or more of (i) one of the amplification oligomers has amodified target hybridizing sequence and the other does not and/or (ii)each of the amplification oligomers has a different unique unpaired 5′sequence; wherein each primer is present in the reaction mixture at adifferent amount P_(x), where x is an integer between 1 and the numberof primers in the mixture; (b) under amplification conditions, eachprimer simultaneously and independently produces one of at least twoamplicons from the target nucleic acid sequence, wherein for eachamplicon, a number of copies A_(x) is produced, and wherein each A_(x)differs by an amount that is approximately same as the amount ofdifference in each P_(x) of the at least two amplification oligomershybridizing to the same strand of the target nucleic acid sequence. Inone aspect, the difference in the A_(x) for each amplicon is at leasttwo orders of magnitude. (c) The at least two amplicons are detectableby hybridization with at least two probes, wherein each of the at leasttwo probes is specific to one of the at least two amplicons, and eachprobe is detectable within a linear detection range C_(x(a)) toC_(x(b)), wherein C_(x(a)) is the minimum detectable number of copies ofamplicon and C_(x(b)) is the maximum detectable number of copies ofamplicon for probe x, wherein for each probe, C_(x+1(a)) is greater thanC_(x(a)) and C_(x+1(b)) is greater than C_(x(b)) such that the at leasttwo probes together are detectable across a wide dynamic range, whereinfor each amplicon-probe combination, A_(x) is between C_(x(a)) andC_(x(b)), and wherein the at least two probes produce distinguishabledetection signals.

In some aspects, the at least two amplification oligomers arepromoter-based amplification oligomers. In some aspects, the at leasttwo amplification oligomers are primers.

The present application further provides reaction mixtures comprising atleast three amplification oligomers, wherein a one of the amplificationoligomers hybridizes to one strand of a target nucleic acid and a secondand a third of the amplification oligomers hybridize to the other strandof a target nucleic acid in order to produce two or more differentiableamplicons. Each species of differentiable amplicon is defined at one endby the common first amplification oligomer. The two amplificationoligomers hybridizing the same strand will define the opposite end oftheir respective amplicon species. In some aspects, the at least threeamplification oligomers are at least three primers. In some aspects, theat least three amplification oligomers are a primer hybridizing to onestrand of a target nucleic acid and two promoter-based oligomershybridizing to the other strand of a target nucleic acid. In someaspects, the at least three amplification oligomers are a promoter basedamplification oligomer hybridizing to one strand of a target nucleicacid and two primers hybridizing to the other strand of a target nucleicacid. In some aspects, the second and a third of the amplificationoligomers hybridizing to the same strand of a target nucleic acid,differ by hybridizing to different sequences on the target nucleic acid.In some aspects, the second and a third of the amplification oligomershybridizing to the same strand of a target nucleic acid, differ from oneanother by having one or more of (i) one of the amplification oligomershas a modified target hybridizing sequence and the other does not and/or(ii) each of the amplification oligomers has a different unique unpaired5′ sequence.

The present application further provides reaction mixtures comprising atleast two detection probe oligomers each of which hybridize to one oftwo or more differentiable amplicons generated using at least threeamplification oligomers, wherein a one of the amplification oligomershybridizes to one strand of a target nucleic acid and a second and athird of the amplification oligomers hybridize to the other strand of atarget nucleic acid. In some aspects, the detectable probe oligomers arelabeled different chemiluminescent labels. In some aspects thedetectable probe oligomers are labeled different fluorescent labels. Insome aspects the detectable probe oligomers are labeled differentfluorescent labels and/or different chemiluminescent labels.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram that illustrates components of in vitroamplification reactions described in Example 1. FIG. 1A depicts thelocations of the promoter-primer (Pr-primer; SEQ ID NO:1), Primer 1 (SEQID NO:2), and Primer 2 (SEQ ID NO:3) relative to a target RNA transcript(SEQ ID NO:4) (described in the “Amplification Compositions” section ofthe Examples). FIG. 1B shows a shorter amplicon (SEQ ID NO:9) and alonger amplicon (SEQ ID NO:10) that result from the reaction of (a) thePr-primer and Primer 1 and (b) the Pr-primer and Primer 2, respectively,and the locations of the two amplicons relative to the sequences in FIG.1A. FIG. 1C depicts the positions of labeled detection probes of SEQ IDNOS:5 and 6 (described in the “Detection Compositions” section of theExamples) relative to the amplicons of FIG. 1B.

FIG. 2 is a graph that shows the mean, background-subtracted, specificactivity-adjusted signal output (y-axis, reported across a range ofabout 10⁴ to about 10⁹ RLU for “relative light units”) detected by usinglinear AE-labeled probes hybridized to amplicons of target RNA across arange of 10² to 10⁹ input target RNA copies (x-axis) as described inExample 1. Results are shown for (a) labeled probes (1 nM; SEQ ID NO:5)mixed with unlabeled oligonucleotides (50 nM; SEQ ID NO:5) (blackcircles); (b) labeled probes (1 nM; SEQ ID NO:6) mixed with unlabeledoligonucleotides (200 nM; SEQ ID NO:6) (black squares); and (c) the sumof detection signals from (a) and (b) (white triangles).

FIG. 3 is a graph that shows the mean, background-subtracted, specificactivity-adjusted signal output (y-axis, reported across a range ofabout 10⁴ to about 10¹¹ RLU) detected by using linear AE-labeled probeshybridized to amplicons of target RNA across a range of 10² to 10⁹ inputtarget RNA copies (x-axis). In this Figure, values obtained in Example 1for the reactions of labeled and unlabeled probes of SEQ ID NO:6 havebeen multiplied by a factor “k” of 46.6 (results shown as black squares)to allow direct visual comparison of these results to those obtainedfrom the reaction of labeled and unlabeled probes of SEQ ID NO:5 (blackcircles). The sum of the two sets of detection signals (white triangles)demonstrate that the reaction system creates a continuous linear dynamicrange of at least 10³ to 10⁹ input copies.

FIG. 4 is a diagram that illustrates the relative alignment of theprimers to the RNA transcript, the resulting amplicons, and the relativealignment of the probes to the amplicons used in Examples 2, 3 and 5. Asin FIG. 1, FIG. 4A depicts the locations of the promoter primer(Pr-primer; SEQ ID NO:1), Primer 1 (SEQ ID NO:2), and Primer 2 (SEQ IDNO:3) relative to a target RNA transcript (SEQ ID NO:4), and FIG. 4Bshows the two amplicons (SEQ ID NOS:9 and 10) which result from thereaction of (a) the Pr-primer and Primer 1 and (b) the Pr-primer andPrimer 2, and the locations of the two amplicons relative to thesequences in FIG. 4A. FIG. 4C depicts the positions of labeled andunlabeled detection probes SEQ ID NO:7 and SEQ ID NO:8 (described in the“Detection Compositions” section of the Examples) relative to theamplicons of FIG. 4B.

FIG. 5 is a graph showing the mean, background-subtracted PMT 1 (lowwavelength) RLU output from reactions of self-quenching AE probes withamplicons of RNA transcripts at the indicated range of copy inputs asobtained in Example 2 for (a) 10 nM labeled probe of SEQ ID NO:7 (blackcircles) and (b) 10 nM labeled probe of SEQ ID NO:7 plus 100 nM labeledprobe of SEQ ID NO:8 (white circles).

FIG. 6 is a graph showing the mean background-subtracted PMT 2 (highwavelength) RLU output from reactions of self-quenching AE probes withamplicons of RNA transcripts at the indicated range of copy inputs asobtained in Example 2. Results are shown for (a) 100 nM labeled probe ofSEQ ID NO:8 (black triangles) and (b) 100 nM labeled probe of SEQ IDNO:8 plus 10 nM labeled probe of SEQ ID NO:7 (white triangles).

FIG. 7 is a graph depicting the mean, background-subtracted RLU outputfrom the reactions of self-quenching AE probes with amplicons of RNAtranscripts at the indicated range of copy inputs as obtained in Example2. Results shown are (a) PMT 1 RLU for 10 nM labeled probe of SEQ IDNO:7 plus 100 nM labeled probe of SEQ ID NO:8 (white circles) and (b)PMT 2 RLU for 10 nM labeled probe of SEQ ID NO:7 plus 100 nM labeledprobe of SEQ ID NO:8 (white triangles).

FIG. 8 is a graph showing the mean, background-subtracted PMT 1 RLUoutput from the reactions of self-quenching AE probes with amplicons ofRNA transcripts at the indicated range of copy inputs as obtained inExample 3. Results shown are for (a) 10 nM labeled probe of SEQ ID NO:7(black circles) and (b) 10 nM labeled probe of SEQ ID NO:7 plus 100 nMlabeled probe of SEQ ID NO:8 (white circles).

FIG. 9 is a graph showing the mean, background-subtracted PMT 2 RLUoutput from the reactions of self-quenching AE probes with amplicons ofRNA transcripts at the indicated range of copy inputs as obtained inExample 3. Results shown are from (a) 100 nM labeled probe of SEQ IDNO:8 (black triangles) and (b) 100 nM labeled probe of SEQ ID NO:8 plus10 nM labeled probe of SEQ ID NO:7 (white triangles).

FIG. 10 is a graph showing the mean, background-subtracted RLU outputfrom the reactions of self-quenching AE probes with amplicons of RNAtranscripts at the indicated range of copy inputs as obtained in Example3. Results shown are (a) PMT1 RLU for 10 nM labeled probe of SEQ ID NO:7plus 100 nM labeled probe of SEQ ID NO:8 (white circles), and (b) PMT 2RLU for 100 nM labeled probe of SEQ ID NO:8 plus 10 nM labeled probe ofSEQ ID NO:7 (white triangles).

FIG. 11 is a graph showing the mean, background-subtracted PMT 1 RLUoutput from the reactions of self-quenching AE probes with amplicons ofRNA transcripts at the indicated range of copy inputs as obtained inExample 5. Results shown are (a) 10 nM labeled probe of SEQ ID NO:7(black circles) and (b) 10 nM labeled probe of SEQ ID NO:7 plus 100 nMlabeled probe of SEQ ID NO:8 (white circles).

FIG. 12 is a graph showing the mean, background-subtracted PMT 2 RLUoutput from the reactions of self-quenching AE probes with amplicons ofRNA transcripts at the indicated range of copy inputs as obtained inExample 5. Results shown are (a) 100 nM labeled probe of SEQ ID NO:8(black triangles) and (b) 100 nM labeled probe of SEQ ID NO:8 plus 10 nMlabeled probe of SEQ ID NO:7 (white triangles).

FIG. 13 is a graph depicting the mean, background-subtracted RLU outputfrom the reactions of self-quenching AE probes with amplicons of RNAtranscripts at the indicated range of copy inputs as obtained in Example5. Results shown are (a) PMT1 RLU for 10 nM labeled probe of SEQ ID NO:7plus 100 nM labeled probe of SEQ ID NO:8 (white circles), and (b) PMT 2RLU for 100 nM labeled probe of SEQ ID NO:8 plus 10 nM labeled probe ofSEQ ID NO:7 (white triangles).

DETAILED DESCRIPTION

As used herein, the terms “including,” “containing,” and “comprising”are used in their open, non-limiting sense.

As used herein, “a” or “an” means “at least one” or “one or more.”

For any quantitative expression used herein, it is understood thequantity is meant to refer to the actual value and is also meant torefer to the approximation of the value that would be inferred by one ofskill in the art, including approximations due to the experimentaland/or measurement conditions for the given value. This inference isintended regardless of whether the term “about” is used explicitly withthe quantity or not.

Target nucleic acids for the present methods and compositions includedouble-stranded and single-stranded targets. Target nucleic acids may befull-length sequences or fragments thereof and may be RNA or DNA. In itsbroadest use, the term target nucleic acids refers to the nucleic acidspresent in the biological sample and the amplified copy thereof. Theamplified copy, may be referred to herein as “amplified copy,”“amplified strand,” “cDNA,” “synthesized complementary sequence,” “RNAtranscript” or other similar term indicating that the referenced strandis not from the biological sample. Under the present method, targetnucleic acid in a test sample may be detected and quantified in amountsranging from 0 to about 10¹² copies.

The term “dynamic range,” as used herein, refers to the ratio betweenthe largest and smallest concentrations or number of copies of a targetnucleic acid that can be detected. In such cases, the dynamic range isused without any designated units. In some circumstances, “dynamicrange” is used to refer to limiting values of detection (e.g., the highand low limits of detection), in which case the appropriate unitmeasure, such as μM or copies, is designated. A “wide” or “broad”dynamic range for the present invention is about 10² to 10¹⁰ in terms ofthe detectable number of copies, or may be range of 10³ to 10⁷ in termsof ratio. For example, a detection method according to the invention mayaccurately detect amounts ranging from about 10³ to 10¹⁰ input targetnucleic acid copies (a dynamic range of about 10⁷), from about 10⁴ to10¹⁰ input sequence copies (a dynamic range of 10⁶), amounts rangingfrom 10³ to 10⁹ input sequence copies (a dynamic range of 10⁶), oramounts ranging from 10⁶ to 10¹⁰ input sequence copies (a dynamic rangeof 10⁴). Thus, in some embodiments, the methods and compositionsprovided herein allow for a detection range from about 10³ to 10⁷ orfrom about 10⁴ to 10⁶. Ranges are understood to be the recited valuesand the whole and partial values in between (e.g. 10² to 10¹⁰ includesthe recited values and values in between such as 10⁵, 2.5×10⁶, 10^(4.5),10^(8.3), etc.).

The term “distinguishable,” as used herein, refers to the characteristicof a nucleic acid sequence that allows it to be detected in a mannerthat distinguishes it from another nucleic acid sequence. For example,two amplicons are distinguishable when they are capable of beingindependently detected by two selective probes. The two probes willgenerate signals that may be detected individually and withoutsignificant interference between them, e.g., through differentwavelengths, different radioligands, or different chemically-detectabledevices (such as a fluorophore and a chemiluminescent probe), or amixture of such signaling mechanisms.

In particular, the term “distinguishable detection signal” refers to thesignals produced by two or more probes. To be “distinguishable,” thesignals produced by the individual probes are differentiable by standarddetection devices. The signals are readily quantifiable by a detectioninstrument without unwarranted interference or overlap between thesignals. For example, probes that produce distinguishable detectionsignals are those that produce signals at wavelengths sufficientlyseparated to be accurately detected and quantified by a detectioninstrument.

The term “amplification oligomer” refers to oligonucleotide sequencesuseful for amplification of a target nucleic acid. Amplificationoligomers include primers, promoter-based amplification oligomers,promoter primers, promoter providers and the like.

The term “promoter-based amplification oligomer” includespromoter-primers and promoter-providers. A “promoter-provider” or“provider” refers to an oligonucleotide comprising first and secondregions, and which is modified to prevent the initiation of DNAsynthesis from its 3′-terminus. The “first region” or “targethybridizing region” of a promoter-provider oligonucleotide comprises abase sequence which hybridizes to a DNA template, where the hybridizingsequence is situated 3′, but not necessarily adjacent to, a promoterregion. The target hybridizing region of a promoter-basedoligonucleotide is typically at least 10 nucleotides in length, and mayextend up to 50 or more nucleotides in length. The “second region” or“promoter region” comprises a promoter sequence for an RNA polymerase. Apromoter provider is engineered so that it is incapable of beingextended by an RNA- or DNA-dependent DNA polymerase, e.g., reversetranscriptase, preferably comprising a blocking moiety at its3′-terminus as described above. As referred to herein, a “T7 Provider”or T7 Promoter Provider is a blocked promoter-provider oligonucleotidethat provides an oligonucleotide sequence that is recognized by T7 RNApolymerase. A similar oligomer that lacks modification to the3′-terminus is called a “promoter primer.” T7 or other promotersequences are useful for promoter-based amplification oligomers. As usedherein, a “promoter” is a specific nucleic acid sequence that isrecognized by a DNA-dependent RNA polymerase (“transcriptase”) as asignal to bind to the nucleic acid and begin the transcription of RNA ata specific site.

As used herein, the term “primer” refers to an oligonucleotide thathybridizes a target nucleic acid and produces an amplicon therefrom.

For some embodiments of the presently disclosed methods andcompositions, at least three amplification oligomers are used together,one amplification oligomer hybridizing one strand and two amplificationoligomers hybridizing the other strand. In any given method orcomposition, at least two amplification oligomers read the targetnucleic acid in the same direction, i.e., either all forwardamplification oligomers or all reverse amplification oligomers. The atleast three amplification oligomers include, then, a forward or reverseamplification oligomer and at least two reverse or forward amplificationoligomers, respectively. The at least two amplification oligomershybridizing the same strand of the target nucleic acid (i.e. samedirection) generate differentiable amplicons by hybridizing two distinctsequences within the target nucleic acid sequence, by hybridizing thesame sequences within the target nucleic acid sequence but by alsoproviding differentiable sequences (modified and unmodified sequences,unique non-hybridizing 5′ sequences or the like). For the same directionamplification oligomers, their target sequences on the target nucleicacid, when distinct, may overlap. The at least two amplificationoligomers are provided in unequal amounts, thereby generating theirrespective differentiable amplicons in approximately the same unequalamounts.

For some embodiments of the presently disclosed methods andcompositions, at least two amplification oligomers are used together,one amplification oligomer hybridizing one strand and one amplificationoligomer hybridizing the other strand. Using this embodiment, thedifferentiable amplicons generated with these amplification oligomersdiffer in their nucleotide composition. For example, using a dNTP mixthat provides one of the dNTPs in an unequal ratio of native dNTP toanalog dNTP will generate unequal amplicons species having the native oranalogue residues. As a further example, using an amplification reactionthat generates RNA amplicon species and DNA amplicon species will alsogenerate unequal amplicons species comprising RNA or DNA.

As used herein, the term “amplicon” refers to the product generated fromthe hybridization of an amplification oligomer with its target sequenceunder amplification conditions. Amplicons can include double strandedDNA, as is generated in PCR amplification reactions and some steps ofisothermal amplification reactions, DNA:RNA hybrids as are generated insome steps of RT-PCR and some steps of isothermal amplificationreactions, or single stranded RNA as are generated in some steps ofisothermal amplification.

As used herein, the term “amplification oligomer-amplicon combination”or “amplification oligomer-amplicon system” refers to an amplificationoligomer and the specific amplicon produced therefrom. The amplificationoligomer referred to in some embodiments is one of the differentiablesame direction amplification oligomers that are described herein forgenerating differentiable amplicons.

As used herein, the term “probe” refers to a DNA or RNA fragment,oligonucleotide or transcript capable of detecting the presence of aparticular nucleotide sequence that is complementary to the sequence inthe probe. Probes are suitably tagged or labeled with a molecular markerthat can be detected by a detection instrument. Suitable probes andinstruments are known in the art. Probes useful in the methods andcompositions described herein may be any probe which allows forquantitative detection of a nucleic acid product. Suitable probesinclude chemiluminescent probes (including self-quenching varieties),fluorophore-based probes, wavelength-detectable probes, radiolabelledprobes, and the like. Particularly useful probes include those labeledwith chemiluminescent labels, such as acridinium esters (AE),hybridization-induced chemiluminescent signal (HICS) labels,fluorophore-AE hybrid labels, and the like. In particular, HICS probesare described in, e.g., U.S. Pat. No. 7,169,554. HICS probes may includewavelength-shifted HICS (wsHICS) probes in which the emission wavelengthis shifted from AE to that of a proximal fluorophore in a self-quenchingprobe format (e.g., U.S. Pat. No. 6,165,800). Thus, self-quenchingenergy transfer probes are examples of suitable probes for use in thepresent methods. Particularly suitable probes include acridinium esterprobes or HICS probes.

Suitable amplification conditions include the amplification methodsdescribed herein. For example, the isothermal amplification or cyclicalamplification methods are appropriate for use in the methods and withthe compositions described herein.

The methods and compositions described herein provide quantitativeamplification of target nucleic acid sequences that may be present in atest sample by using various means to produce different quantities ofdistinguishable detectable amplicon products in known proportions. Thedescribed methods and compositions generate multiple differentiableamplicons at ratios relative to each other and relative to the amount oftarget nucleic acid sequence. Amplification of a target nucleic acidsequence with a single pair of amplification oligomer could generate anamplicon with a concentration outside the accurate detection range of aprobe or the detection instrument. According to the present invention,in contrast, multiple amplification oligomer-amplicon systems are usedto simultaneously generate multiple differentiable amplicons atdiffering amplicon concentrations. Thus, rather than one narrow windowof accurate detection, the present method creates multiple overlappingdetection ranges, each of which is accurate for a particular species ofamplicon product. The present method produces multiple differentiableamplicons in a single amplification system, which is preferablyperformed in a single vessel. The differentiable amplicons may differ inlength, wherein the amplicon species share a common sequence, butwherein each longer amplicon species adds additional sequence that isunique to the longer species compared to the shorter. The differentiableamplicons may differ in composition, wherein one or more modifiednucleobases in incorporated into a species of amplicon, but not intoother(s). Similarly, the differentiable amplicons may differ incomposition, wherein separately unique 5′ sequences are incorporatedinto each species of amplicon by using amplification oligomers with 5′non-hybridizing sequences. The differentiable amplicons may differ incomposition, wherein a modified nucleobase is combined with anunmodified nucleobase for incorporation into a subset of amplicons(e.g., a dNTP mix having a ratio of dATP and modified dATP or asubstitute for dATP).

In these methods, the number of differentiable amplicons and their fixedratios relative to one another are chosen to span the required dynamicrange for the amplification reaction and to accommodate limitations ofthe measuring system that detects the amount of the amplicons generatedin the amplification reaction. In essence, the amplicons are surrogatemarkers for the target nucleic acid sequence and appear (and are thusdetectable) at a wide range of amounts rather than the single amount ofthe target sequence. For example, the compositions and conditions areselected to generate a large amount of amplicon 1 and a relativelysmaller amount of amplicon 2 (and, optionally, a still smaller amount ofamplicon 3, and etc for each amplicon species made to extend the dynamicrange of detection) in a single reaction mixture. If the amount ofamplicon 1 produced in the amplification reaction exceeds the dynamicrange of the detection system, the amounts of amplicon 2 (and/oramplicon 3) produced may be detected instead, as their concentrationsfall within the dynamic range of the system. In some embodiments, thenumber of copies of each amplicon species differs by two, three, four ormore orders of magnitude.

The amplicons and amounts thereof are selected to ensure that one ormore of the amplicons made in the amplification reaction mixture may beprecisely measured in the detection system. From the precise ampliconmeasurements, the amount of original target nucleic acid sequencepresent in the test sample is calculated. Overall, then, the system iscapable of accurately detecting and quantifying the target nucleic acidsequence over an extended dynamic range. In a preferred embodiment,different amounts of amplification oligomers that target the same strandof the target nucleic acid are used to produce the different amounts ofdistinguishable amplicons. In some embodiments, the amount of eachamplification oligomer differs by at least two orders of magnitude. Insome embodiments, the distinguishable amplicons are made from the sametarget region of the target nucleic acid sequence. In other embodiments,the distinguishable amplicons are made from overlapping target regions.In yet other embodiments, the distinguishable amplicons are made fromdistinct target regions.

In some embodiments, the starting target amount is determined using oneor more equations that include terms containing the concentration ofeach primer or terms that express the relative relationship among theconcentrations of the primers, such as a ratio, that lead to theformation of a distinguishable product. Relevant variables for thecalculation include the concentrations of the various amplificationoligomers and their amplification efficiencies.

In other embodiments, the methods use primers selected to producedistinguishable amplicons in which the amounts of those primers arechosen to stop amplification of each distinguishable amplicon at apreselected level that cannot exceed the amounts of the relevant primersfor that amplicon.

The actual ratios at which the distinguishable amplification oligomertargeting the same strand of a target nucleic acid are present in thereaction will depend upon the inherent dynamic range of the detectionsystem. The ranges of the amounts of amplicon produced by each primermay ideally overlap somewhat to produce an unbroken extended dynamicrange unless there are reasons that separate dynamic ranges with a gapbetween them is desired. For example, if acridinium ester-labeled probesand a luminometer capable of detecting amplicon over a span of 4 logsare used in a system in which two distinguishable products are made,then the ratio of the primers used to make the two distinguishableproducts should be between 1,000 and 10,000 and, more preferably, about3000, the midpoint of the logarithmic range between those two extremes.

The methods of the invention may be described according to the followingexemplary scheme. Although the scheme is depicted with just twoamplification oligomer-amplicon systems, one of skill in the art willrecognize that additional amplification oligomer-amplicon-probe systemsmay be added to create an even broader dynamic range. It is also notablethat the schematic below showing the two amplification oligomer-ampliconsystem is not showing the opposite strand amplification oligomer thatworks with P1 and P2 to generate A1 and A2. The variables P_(x), A_(x),and C_(x) are defined above.

As discussed above, the detection ranges for the individual probes maybe discreet (i.e., a gap between the ranges) or overlapping. The schemeabove depicts the overlapping variant. Thus, in particular embodiments,each C_(x(b)) is greater than C_(x+1(a)) such that the ranges ofdetection for the probes overlap, creating a continuous dynamic range.

As used herein, the variable T₁ refers to the number of copies of targetnucleic acid sequence in a test sample. T₁ falls within a range fromabout T_(a) to T_(b), wherein the range represents the amount of targetnucleic acid sequence that will produce an amount of amplicon that fallswithin the dynamic detection range of the system. For example, accordingto the Scheme above, for a given T₁, the amounts of AmplificationOligomer 1 (P₁) and Amplification Oligomer 2 (P2) are selected such thatthe resulting amounts Amplicon 1 (A₁) and Amplicon 2 (A2) fall withinthe C_(1(a)) to C_(2(b)) range of detection for the system.

Distinguishable Products Made Using Modified and Unmodified Primers

In one embodiment of the method, the amplification oligomers generateamplicon species that differ one from the other by the presence orabsence of a modified nucleobase. In one aspect, the amplificationoligomers targeting the same strand of a target nucleic acid differ fromone another by having one or more modified nucleobases in the targethybridizing sequence of one amplification oligomer, compared to another.The modified and unmodified amplification oligomers can hybridize to thesame sequence on the same strand of a target nucleic acid, yet producedifferentiable amplicons based on the incorporation of the modifiednucleobase in some of the amplification products. For example, a TMAreaction is run using a single promoter-based amplification oligomer andtwo primer oligomers, wherein the one of the two primers is anunmodified primer and the other is a modified primer. A modified primeris derived from the sequence of the unmodified primer to contain adistinguishable modification that is incorporated into its amplicon. Ina preferred embodiment of the modified primer, one or more of thenucleotides in the unmodified primer are replaced by other nucleotides.In an especially preferred embodiment, the substitution does notsubstantially alter the ability of the modified primer to anneal to itsintended target nucleic acid sequences and promote amplificationrelative to the unmodified primer. The amplification reaction is thenrun using the unmodified primer and a small fraction, for example1/1000, of the modified primer. Thus, the amplification reaction willgenerate a first amplicon corresponding to the modified primer and asecond amplicon corresponding to the unmodified primer. Assuming themodified and unmodified primers are equally efficient, the number offirst amplicon molecules with the modified sequence will be about 1/1000of the number of second amplicon molecules with the unmodified sequence.The amplicon populations are then detected and quantified using twodistinguishable probes. One probe detects the second amplicon moleculesthat contain the unmodified sequence; the other probe detects the firstamplicon molecules that contain the modified sequence. If littleamplicon is produced due to low initial target levels, only the probecomplementary to the unmodified sequence will show significanthybridization because this amplicon is produced in excess over themodified amplicon species. This hybridization should be quantitativeover about 3-4 logs. If, however, the amount of amplicon produced in theamplification reaction is much greater, then the probe that iscomplementary to the unmodified sequence will reach hybridizationsaturation and signal will be detected with the probe complementary tothe modified sequence. The latter signal will also be quantifiable overabout 3-4 logs; however, to determine the amount of amplicon generatedthat signal needs to be multiplied by 1000 (the “ratio factor”) since itrepresents only 1/1000 of the number of amplicon molecules that havebeen produced in the amplification reaction.

Ideally, the distinguishable products will be produced in amountsequivalent to the ratios of the primers used in their synthesis, thoseskilled in the art however will recognize that exactly matchingamplification efficiencies of different primers may be difficult andexpensive. In other preferred embodiments of the invention, theefficiencies of amplification of the distinguishable products arematched to the degree desired and any difference in efficiencies istaken into account when the total amount of amplicon produced iscalculated from the amounts of each distinguishable product. In theprevious example in which the modified primer was present in 1000-foldsmaller amount than the unmodified primer but amplification efficiencieswere the same, the amount of modified amplicon was multiplied by 1000.If, however, the efficiency of amplification with the modified primerwere to be only 80% of the efficiency of the unmodified primer, thenonly 0.8 molecules of modified product would be synthesized for each1000 molecules of unmodified product. In that case, the amount of totalamplified product would be calculated by multiplying the signal from themodified product by the ratio factor (1000) and by an “efficiencyfactor” (expected/actual=1.0/0.8=1.25) to take into account the lowerefficiency.

For reactions in which hybridization is measured by the number ofrelative light units (RLU) produced from a chemiluminescent label, theamount of total amplicon would be calculated as a function of the signalfrom each probe as follows:Total amplicon(probe U signal)=Net RLU_(probe U)×(pmoles probeU/RLU_(probe U))  (1);Total amplicon(probe M signal)=Net RLU_(probe M)×(pmoles probeU/RLU_(probe M))×ratio factor×efficiency factor  (2)

where “probe U” and “probe M” target the unmodified and modifiedamplicons, respectively.

If both calculated values lie within the overlap region of the dynamicrange, then in a preferred embodiment they are compared to determine ifthey are acceptably close to one another. If they do not differ by morethan a predetermined acceptable amount, then they are combined toproduce the best estimate of the total amplicon amount. In general, themean value will be determined by summing the two values and dividing by2.

In general, where the amount of amplicon produced is above the overlaprange, the signal from probe M will be used to calculate the totalamount of amplicon. In this case, the signal from probe U is expected tobe saturated. In the case where the amount of amplicon produced is belowthe overlap range, the signal from probe U will be used to calculate thetotal amount of amplicon. In this case, the signal from probe M isexpected to be less than the signal from probe U. How much less willdepend upon the ratio factor, the efficiency factor, and the specificactivities of the two probes as well as the amount of target.

The amount of target nucleic acid sequence in the test sample is thendetermined in comparison to values of total amplicon produced under thesame reaction conditions from a set of standards of known target nucleicacid sequence concentration.

In the example discussed above, two distinguishable products wereproduced and the amount of target nucleic acid sequence in the testsample was calculated based upon the amounts of each of the twoamplicons generated in the amplification reaction. Those skilled in theart will appreciate that further increases in the dynamic range of anassay can be achieved by making additional numbers of distinguishableamplicons in the amplification reaction through the use of additionalprimers as needed to achieve the desired dynamic range (e.g., P₃, P₄,etc.). Each new amplicon should be distinguishable from the others andbe made in an amount that generates a signal within the range of thedetection instrument when it is hybridized to a probe with anappropriate specific activity. The amount of primer used to generateeach specific amplicon must result in an amount of product thatcontributes to the extension of the dynamic range. In particularembodiments, as discussed earlier for the case in which twodistinguishable amplicons are used, the amount of product produced fromeach added primer will fall within a detection range that partially, butnot completely, overlaps the detection range for product produced by anyof the other primers in order to extend the dynamic range beyond thelimits it had without the new primer.

In a preferred embodiment, the specific activities of the probes aresimilar or identical and the dynamic range is spanned by differences inthe amounts of each distinguishable product that is made. However, inother embodiments both the amounts of distinguishable products made andthe specific activities of the probes used to detect them may be variedto further expand the dynamic range or simply for convenience. Thespecific activity of each probe (that is, the amount of signal per molaramount of probe) is a term of the equations given above to calculate theamount of each distinguishable amplicon that is produced. In practice,it may be difficult to achieve similar or identical specific activitieswhen labeling probes; therefore, the ability of the invention toaccommodate some variability in specific activity is often of value.

As noted above, the modified primer may contain one or more basesubstitutions. These may be located at different positions within theprimer with intervening unsubstituted bases or they may be clusteredtogether in a single region. A sufficient number of nucleotides may besubstituted to form a region that loops out when the primer hybridizesto the target nucleic acid sequence. Any of a large number ofmodifications is possible so long as they do not substantially alter theefficiency of priming and amplification. By “not substantially alter” ismeant that the base substitutions do not change the efficiency ofamplification so that the amounts of product produced from them arebelow the levels needed to span the intended dynamic range in accordancewith the teachings herein of the invention or cause the amount ofproduct synthesized from them to be excessively variable under thereaction conditions and with the target nucleic acid sequences and testsample types used. By “excessively variable” is meant that the amount ofamplicon produced cannot be held within the desired range of performanceneeded when the assay is applied for its intended use. In the case of anassay intended for measurement of viral loads in patient blood samples,the coefficient of variation of the assay should generally be no morethan about 25% and preferably less than about 10%.

The modified and unmodified amplicons could either be detected usingprobes that can distinguish between the two forms, or an enzyme thatdistinguishes the two could be used to help effect discrimination. Forexample, if the modified base is a methylated base, then a restrictionenzyme that does not recognize the methylated sequence might beemployed. Alternately, for example, if the modified base is a methylatedbase, then a restriction enzyme that does recognize the methylatedsequence might be employed. Alternatively, the presence of the modifiedbase may cause a different base to be inserted in the amplicon on thenext round of replication. In this case, the base could be adeoxyribonucleotide or a ribonucleotide. The modified sequence that isproduced could be differentiated from the unmodified sequence using twodistinguishable probes that each hybridize to only one of the two forms.

Distinguishable Products Made Using Primers with 5′ Unpaired Bases

Another method for providing distinguishable amplicons involves usingsame direction amplification oligomers with 5′ unpaired bases. Forexample, two primers might each contain one or more originally unpairedbases at or near their 5′ termini, wherein the unpaired bases of oneamplification oligomer is different from the unpaired bases of the otheramplification oligomer. By “originally unpaired” is meant that thesebases are not hybridized when the primer anneals to the target nucleicacid sequence in the test sample. Once the primer and its 5′ unpairedbase is incorporated into an amplicon as the amplification reactionproceeds, then each primer will be completely complementary to thoseamplicon molecules that are derived from the same primer sequence butwill not be completely complementary to those amplicon molecules thatwere derived from the other primer sequence. These two forms of primerare likely to have similar kinetics with respect to amplification.

Distinguishable Products Made Using Substituted Nucleotides

In another embodiment of the method, one of the nucleotide triphosphates(NTPs) present in the amplification reaction is replaced in part with amodified NTP analog with different hybridization or other recognitioncharacteristics once incorporated into the amplicons. The modified NTPsare present at, for example, about 1/1000 of the numbers of theunmodified NTPs. As amplicon is produced, the sequence complementary tothe detection probe will contain, on average, 1 molecule with themodified base in a particular position instead of the unmodified baseper 1000 nucleotides incorporated. The modified base could either bedetected using probes that can distinguish between the two forms, or anenzyme that distinguishes the two could be used to help effectdiscrimination. For example, if the modified base is a methylated base,then a restriction enzyme that does not recognize the methylatedsequence might be employed. Alternately, a probe could have a modifiednucleotide at a position that is complementary to the modifiednucleotide incorporated into a fraction of the amplicons but notcomplementary to other nucleotides incorporated in that position.Modified nucleotides, as well as enzymatic incorporation of modifiednucleotide triphosphates, are known in the art (e.g., Nucleic Acids Res.26(21): 4975-4982 (1998); J. Am. Chem. Soc. 122(32): 7621-7632 (2000);Proc. Natl. Acad. Sci. U.S.A. 100(8): 4469-4473 (2003); J. Am. Chem.Soc. 125(33): 9970-9982 (2003); J. Am. Chem. Soc. 126(4): 1102-1109(2004); J. Am. Chem. Soc. 127(43): 15071-15082 (2005)). Alternatively,the presence of the modified base may cause a different base to beinserted in the amplicon on the next round of replication. In this case,the base could be a deoxyribonucleotide or a ribonucleotide. Themodified sequence that is produced could be differentiated from theunmodified sequence using two distinguishable probes that each hybridizeto only one of the two forms.

Distinguishable DNA and RNA Targets

Certain amplification systems inherently produce different amounts ofdistinguishable amplicons as part of the amplification process. In oneembodiment of the method, differential detection of the final andintermediate products of these amplification reactions is used to extendthe dynamic range. For example, in a commonly practiced variant of TMA,the most abundant amplicon is RNA that is complementary to the targetnucleic acid. Double-stranded DNA is also produced in much smalleramounts, generally at levels that are about 100- to 1000-fold less.Probes can be made to detect the negative strand RNA amplicon speciesand either, or both, strands of the DNA amplicon species. Thus, thegeneral principle of the invention, namely the detection of multipleforms of amplicon that are present in substantially different numbers towiden the dynamic range, can be applied provided that the ratio ofnegative strand RNA amplicon to positive strand DNA amplicon is constantunder the conditions used to conduct the TMA reaction. The relativeamounts of RNA and double-stranded DNA products that are produced in aTMA reaction can be varied by changing the sequence of the promoter usedas described, for example, in Nucleic Acids Res. 15(13): 5413-5432(1987), Nucleic Acids Res. 15(21): 8783-8798 (1987), Nucleic Acids Res.20(10): 2517-2524 (1992), Nucleic Acids Res. 24(18): 3659-3660 (1996),Pac. Symp. Biocomput. 15: 433-443 (2010), Biochemistry 41(11): 3586-3595(2002) and J. Biol. Chem. 280(49): 40707-40713 (2005). Sincetranscription efficiency is also dependent upon the concentration ofribonucleotides, it can be varied by changing both the totalconcentration of these reaction components as well as the relativeamounts of each of the four ribonucleotides (Science 278(5346):2092-2097 (1997); J. Mol. Biol. 281(5): 777-792 (1998)). The activity ofreverse transcriptase in copying both the RNA and DNA templates in thereaction can be varied similarly by altering the concentrations ofdeoxyribonucleotides as well as the relative amounts of each of the fourdeoxyribonucleotides. Reducing the concentrations of all fourdeoxyribonucleotides will slow the reverse transcriptase DNA polymerasereaction relative to the RNA polymerase reaction. For specific targetregions, analysis of the percentages of each base in the DNA and RNAcomponents of the reaction may be used to select concentrations of eachbase that will increase or decrease the rate of synthesis of either thetranscribed RNA or the double-stranded DNA. For example, if an ampliconcontains a high percentage of cytosine nucleotides relative to anotheramplicon, lowering the concentration of cytosine in the reaction mixturecan be used to slow synthesis of that particular amplicon. Alterationsin other reaction parameters such as those described, for example, inU.S. Pat. Nos. 5,705,365 and 5,710,029 can also be used todifferentially affect the ratio of synthesis of transcribed RNA anddouble-stranded DNA products. Detection of the positive strand DNA iscomplicated by the possible presence of competing negative strand RNAamplicon in substantially larger amounts; therefore, the negative strandDNA may be a preferable target. Since the DNA amplicons in TMA containthe promoter sequence, modifications of the promoter-primer that arereadily detectable may be preferred. A probe may be used to confirm thepresence of the promoter region, the adjacent primer region, and aportion of the desired target sequence in the amplicon. These threesequences will only be present in tandem in DNA intermediates in thereaction.

Likewise, in another commonly practiced variant of TMA, single-primerTMA described in U.S. Pat. No. 7,374,885, the most abundant amplicon isRNA that is the same sense as the target nucleic acid. Negative strandDNA is also produced in much smaller amounts. Probes can be made todetect the positive strand RNA and the negative strand DNA to widen thedynamic range.

Distinguishable Products Based Upon Differential Target Capture

In another embodiment of the method, suitable target captureoligonucleotides are made that are complementary or partiallycomplementary to one of the primer sites and extend into a part of thedesired probe-binding site. These target capture oligomers are able toserve as both target capture oligonucleotides and same directionamplification oligomers. In a small percentage of these target captureoligonucleotides, the sequence is altered so that a distinguishableamplicon is produced in the probe-binding region in the same way asdescribed above for a primer that does not also function in targetcapture. After target capture and washing to remove unwanted materials,including unbound capture probe, the remaining components of theamplification reaction are added and the amplification process permittedto occur. The initial DNA:RNA (original target RNA) or DNA:DNA (originaltarget DNA) duplexes that are generated by extension of the targetcapture oligonucleotides will contain the modified sequence in a knownproportion of the molecules. Assuming that the two target sequencesamplify at a consistent ratio thereafter, probes that distinguishbetween the two species can be used to quantitate the original targetover a wider range as discussed above.

Distinguishable Products Made Using Semi-Nested Primers

In yet another embodiment of the method, semi-nested primers areprovided in an amplification reaction at different concentrations. Forexample, the at least two primers comprise a first inner primer and asecond outer primer. The first inner and second outer primers are bothforward primers or are both reverse primers. The reaction mixture mayfurther comprise a single promoter-based amplification oligomer thatoperates in a direction opposite that of the first inner and secondouter primers. Thus, the at least two amplicons produced from theseprimers comprise a first amplicon produced from the first inner primerand a second amplicon produced from the second outer primer. The firstinner primer is provided at a low concentration while the second outer,reverse primer is provided at a higher concentration. When targetnucleic acid sequences are included in the reactions, shorter ampliconsare synthesized from the promoter-based amplification oligomer and firstinner primer while longer amplicons are synthesized from thepromoter-based amplification oligomer and the second outer primer. Thelonger amplicons can serve as templates for further synthesis of longeror shorter amplicons. By choosing different reverse primerconcentrations, differential amounts of the longer and shorter ampliconsare synthesized in a single reaction. The shorter amplicons includesequences that are in common with the longer amplicons. The longeramplicons include sequences that are in common with the shorteramplicons and others that are different than those in the shorteramplicons. Because differential amounts of longer and shorter ampliconsare synthesized in a single reaction, quantifying the amounts of longerand shorter amplicons can provide two overlapping ranges, effectivelyextending the dynamic range of detection. Quantification of differentamounts of amplicons can be further brought into dynamic range of adetection system by adjustment of probe specific activity.

Alternate configurations of semi-nested amplification oligomers atdifferent concentrations are possible in an amplification reaction atdifferent concentrations. For example, opposite a single reverse primer,an inner forward primer is provided at a low concentration while anouter forward primer is provided at a higher concentration. When targetnucleic acid sequences are included in the reactions, shorter ampliconsare synthesized from the reverse primer and inner forward primer whilelonger amplicons are synthesized from the reverse primer and the outerforward primer. The longer amplicons can serve as templates for furthersynthesis of longer or shorter amplicons. From this process,differential amounts of longer and shorter amplicons are synthesized ina single reaction. Choosing different forward primer concentrations canvary these differential amounts of amplicons. The shorter ampliconsinclude sequences that are in common with the longer amplicons. Thelonger amplicons include sequences that are in common with the shorteramplicons and others that are different than those in the shorteramplicons. Since differential amounts of longer and shorter ampliconsare synthesized in a single reaction, quantifying the amounts of longerand shorter amplicons can provide two overlapping ranges, effectivelyextending the dynamic range of detection. Quantification of differentamounts of amplicons can be further brought into dynamic range of adetection system by adjustment of probe specific activity.

The following examples are provided to further illustrate the presentmethod and are not intended to limit to it.

EXAMPLES Amplification Compositions for Examples 1-3 and 5

0.2 μm filtered water (H₂O)

4× Amplification Reagent: 160 mM tris(hydroxymethyl)aminomethane (Trisbase), 100 mM MgCl2, 70 mM KCl, 16 mM each of rATP, rCTP, rGTP and rUTP,4 mM each of dATP, dCTP, dGTP and dTTP, 20% polyvinylpyrrolidone, 0.3%ethyl alcohol, 0.02% methyl paraben, 0.01% propyl paraben, pH 7.5

4× Enzyme Reagent: 8 mMN-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES freeacid), 140 mM Tris base, 70 mM KCl, 50 mM N-acetyl-L-cysteine, 1.04 mMethylenediaminetetraacetic acid (EDTA), 0.04 mM zinc acetate, 10% TRITONX®-102, 80 mM trehalose, 20% glycerol, 80 units/μL Moloney murineleukemia virus (MMLV) reverse transcriptase (RT), 80 units/μL T7 RNApolymerase (T7RNAP), pH 8.0

Silicone Oil Reagent: polydimethylsiloxane, trimethylsiloxy terminated

Primers:

4× (Promoter primer or Pr-primer (300 nM in water)

(SEQ ID NO: 1) 5′-AATTTAATACGACTCACTATAGGGAGAGTTTGTATGTCTGTTGCTAT TAT-3′

4× Primer 1 (15 nM in water)

(SEQ ID NO: 2) 5′-ACAGCAGTACAAATGGCAG-3′

4× Primer 2 (285 nM in water)

(SEQ ID NO: 3) 5′-ATTCCCTACAATCCCCAAAGTCAA-3′

Target sequence—RNA, 1,016 nt, in vitro transcript (IVT); the 51 nt fromthe 5′ part of the IVT are from the T7 promoter and cloning vector; theremaining 965 nt include the 3′ part of the HIV-1 subtype B pol gene,possibly a little of the 5′ part of the subsequent gene depending on whoassigned the division of genes in water

(SEQ ID NO: 4) 5′-GGGAGACAAGCUUGCAUGCCUGCAGGUCGACUCUAGAGGAUCCCCGGGUACCAGCACACAAAGGAAUUGGAGGAAAUGAACAAGUAGAUAAAUUAGUCAGUGCUGGAAUCAGGAAAAUACUAUUUUUAGAUGGAAUAGAUAAGGCCCAAGAUGAACAUGAGAAAUAUCACAGUAAUUGGAGAGCAAUGGCUAGUGAUUUUAACCUGCCACCUGUAGUAGCAAAAGAAAUAGUAGCCAGCUGUGAUAAAUGUCAGCUAAAAGGAGAAGCCAUGCAUGGACAAGUAGACUGUAGUCCAGGAAUAUGGCAACUAGAUUGUACACAUUUAGAAGGAAAAGUUAUCCUGGUAGCAGUUCAUGUAGCCAGUGGAUAUAUAGAAGCAGAAGUUAUUCCAGCAGAAACAGGGCAGGAAACAGCAUAUUUUCUUUUAAAAUUAGCAGGAAGAUGGCCAGUAAAAACAAUACAUACAGACAAUGGCAGCAAUUUCACCAGUGCUACGGUUAAGGCCGCCUGUUGGUGGGCGGGAAUCAAGCAGGAAUUUGGAAUUCCCUACAAUCCCCAAAGUCAAGGAGUAGUAGAAUCUAUGAAUAAAGAAUUAAAGAAAAUUAUAGGACAGGUAAGAGAUCAGGCUGAACAUCUUAAGACAGCAGUACAAAUGGCAGUAUUCAUCCACAAUUUUAAAAGAAAAGGGGGGAUUGGGGGGUACAGUGCAGGGGAAAGAAUAGUAGACAUAAUAGCAACAGACAUACAAACUAAAGAAUUACAAAAACAAAUUACAAAAAUUCAAAAUUUUCGGGUUUAUUACAGGGACAGCAGAAAUCCACUUUGGAAAGGACCAGCAAAGCUCCUCUGGAAAGGUGAAGGGGCAGUAGUAAUACAAGAUAAUAGUGACAUAAAAGUAGUGCCAAGAAGAAAAGCAAAGAUCAUUAGGGAUUAUGGAAAACAGAUGGCAGGUGAUGAUUGUGUGGCAAGUAGACAGGAUGAGGAUUAGAACAUGGAAAAGUUUAGUAAAACACCA-3′

Detection Compositions for Examples 1-3 and 5

Hybridization Reagent: 100 mM succinic acid, 2% (w/v) lithium laurylsulfate (LLS), 230 mM LiOH, 15 mM Aldrithiol-2, 1.2 M LiCl, 20 mMethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid(EGTA), 20 mM EDTA, 3% (v/v) ethyl alcohol, pH 4.7

Hybridization Diluent: 0.1% (w/v) LLS, 10 mM succinic acid, pH 5

Alkaline Reagent: 600 mM boric acid, 182 mM NaOH, 1% TRITON X®-100, pH8.5

TMA Separation Suspension Reagent: 0.25 μg/μL magnetic particles (BioMagM4100), 0.2 mM EDTA, 0.08% (w/v) LLS, 8 mM succinic acid, pH 5

Separation Reagent: 1.25 μg/μL magnetic particles (BioMag M4100), 1 mMEDTA

Read Reagent: 125 mM LiOH, 1.5 mM EGTA, 1.5 mM EDTA, 95 mM succinicacid, 8.5% (w/v) LLS, pH 5.2

Detection Reagents

Detection Reagent 1: 32 mM H₂O₂, 1 mM HNO₃

Detection Reagent 2: 1.5 M NaOH

Detection Reagent 3: 240 mM H₂O₂, 1 mM HNO₃

Detection Reagent 4: 2 M Tris base, pH 9.0 with HCl

Detection Reagent 5: 1 mM HNO₃

Probes:

acridinium ester (AE)-labeled linear oligonucleotide probe 1

(SEQ ID NO: 5) 5′-CCACAAUUUUAAAAGAAAAGGG-3′

AE-labeled linear oligonucleotide probe 2

(SEQ ID NO: 6) 5′-AGAAAAUUAUAGGACAGGUAAG-3′

unlabeled oligonucleotide probe 3

(SEQ ID NO: 5) 5′-CCACAAUUUUAAAAGAAAAGGG-3′

unlabeled oligonucleotide probe 4

(SEQ ID NO: 6) 5′-AGAAAAUUAUAGGACAGGUAAG-3′

AE-labeled, Hybridization Induced Chemiluminescent Signal (HICS) probe 5with a 5′-AE and a 3′-[4-(dimethylamino)azobenzene-4′-carboxylic acid](dabcyl) moiety

(SEQ ID NO: 7) 5′-C6amine(LiAE)-CUCGUCCACAAUUUUAAAAGAAAAGGGACG AG-D-3′

AE-labeled, wavelength-shifted Hybridization Induced ChemiluminescentSignal (wsHICS) probe 6 with a 5′-tetramethylrhodamine (TAMRA),5′-penultimate AE and a 3′-dabcyl moiety

(SEQ ID NO: 8) 5′-TAMRA-rxl(LiAE)-CCuCUAGAAAAUUAUAGGACAGGUAAGAGAG G-D-3′

Amplification Methods for Examples

Replicate TMA reactions were performed in Examples 1-3 and 5 as follows:25 μL volumes each of 4× Amplification Reagent, primers in water, andRNA transcripts in water plus 100 μL volumes of Silicone Oil Reagentwere mixed and incubated at 60° C. for 10 minutes then at 42° C. for 5minutes; 25 μL volumes of 4× Enzyme Reagent were added to the solutions,the solutions were mixed and then incubated at 42° C. for 90 minutes.The relative placement of the Pr-primer and Primers 1 and 2 on the RNAtranscript and the resulting amplicons are shown diagrammatically inFIGS. 1 and 4. Because different concentrations of Primers 1 and 2 wereused, different amounts of the respective amplicons and probe bindingregions were synthesized in a single reaction.

Example 1: Quantitative Detection of Amplicons with AE Probes Over aDynamic Range in Separate Reaction Chambers

This example demonstrates quantitative detection of amplicons over arange of 10³ to 10⁹ copies of target nucleic acid in separate reactiontubes.

At the completion of the TMA amplification reactions, 100 μL volumes ofprobes in Hybridization Reagent were added to the amplification reactionmixtures, which were mixed and incubated at 60° C. for 15 minutes toallow hybridization of probes specific for complementary sequences inthe synthesized amplicons. The probes used were: (a) AE-labeled probe ofSEQ ID NO:5 (1 nM) plus unlabeled probe of SEQ ID NO:5 (50 nM), whichwas used to attenuate the specific activity of the labeled probe; or (b)AE-labeled probe of SEQ ID NO:6 (1 nM) plus unlabeled probe of SEQ IDNO:6 (200 nM), which was used to attenuate the specific activity of thelabeled probe. The relative placement of the probes on thedistinguishable amplicons is shown diagrammatically in FIG. 1. In thisexample, the AE-labeled probe of SEQ ID NO:5 is able to detect bothamplicons, whereas the AE-labeled probe of SEQ ID NO:6 is only able todetect the longer amplicon.

The initial specific activities for the AE-labeled probes of SEQ ID NO:5and SEQ ID NO:6 were about 1.1×10⁸ and 1.6×10⁸ RLU/pmol, respectively.The specific activities were adjusted by adding various amounts ofunlabeled oligonucleotides of the same sequence as the labeled probe tobalance the dynamic range of the probes and the amplicon output. Theamount of amplicon produced by the end of the amplification reaction wasempirically determined by titrating each detection region with itsrespective probe plus increasing amounts of unlabeled probe until thehighest amount of amplicon output was slightly less than the amount oftotal probe. For the probe of SEQ ID NO:6, 20 pmol total probe/100 μLHybridization Reagent (200 nM with specific activity=8×10⁵ RLU/pmol) wasneeded to exceed amplicon from 10⁹ copies input. For probe of SEQ IDNO:5, 5 pmol total probe/100 μL Hybridization Reagent (50 nM withspecific activity=2.2×10⁶ RLU/pmol) was needed to exceed amplicon from10⁵ copies input.

At the completion of the hybridization reactions, 300 μL volumes ofAlkaline Reagent were added to the hybridization reactions, and theresulting mixtures were incubated at 60° C. for 20 minutes toselectively hydrolyze AE-labeled probes that were not hybridized tocomplementary amplicons.

Chemiluminescence from these reactions was initiated in a GEN-PROBE®LEADER® HC+Luminometer with addition of 200 μL Detection Reagent 1, atwo second pause prior to addition of 200 μL Detection Reagent 2, a 0.04second pause, then acquisition of 50×0.04 second intervals with nodelays between intervals. The chemiluminescent output in relative lightunits (RLUs) from these intervals was summed and the mean values fromreplicates was calculated (Tables 1 and 2).

TABLE 1 1 nM AE-labeled probe of SEQ ID NO: 5 + 50 nM unlabeled probe ofSEQ ID NO: 5 specific activity- copies mean RLU − adjusted, mean inputmean RLU bkgd RLU − bkgd  0 4,677 10² 5,625 948 47,388 10³ 13,639 8,962448,100 10⁴ 64,087 59,410 2,970,500 10⁵ 679,369 674,691 33,734,563 10⁶1,508,029 1,503,351 75,167,563 10⁷ 1,606,846 1,602,169 80,108,438 10⁸1,645,605 1,640,928 82,046,375 10⁹ 1,715,148 1,710,471 85,523,538

TABLE 2 1 nM AE-labeled probe of SEQ ID NO: 6 + 200 nM unlabeled probeof SEQ ID NO: 6 specific activity- specific activity- adjusted, meancopies mean mean RLU − adjusted, mean RLU − bkgd, input RLU bkgd RLU −bkgd x k = 46.6  0 6,963 10² 6,667 10³ 6,859 10⁴ 7,912 949 189,7508,842,350 10⁵ 10,583 3,621 724,100 33,743,060 10⁶ 34,004 27,0415,408,150 252,019,790 10⁷ 180,227 173,264 34,652,800 1,614,820,480 10⁸956,357 1,496,352 299,270,400 13,946,000,640 10⁹ 7,175,753 7,168,7901,433,757,950 66,813,120,470

The mean RLU from the zero RNA transcript input (first entry in eachtable) was used as the background (“bkgd”) measurement and wassubtracted from the results of experiments containing 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸ and 10⁹ copies RNA transcripts input (mean RLU minusbkgd). The mean background-subtracted values (mean RLU−bkgd) wereadjusted to account for the specific activity of the linear AE-labeledprobes by multiplying by the fold excess of the linear probes withoutAE, resulting in specific activity-adjusted, mean background-subtractedvalues (specific activity-adjusted, mean RLU−bkgd). The specificactivity-adjusted, mean background-subtracted values for the two probesmeasured from separate tubes were added, leading to summed specificactivity-adjusted, mean background-subtracted values (Table 3, specificactivity-adjusted, mean RLU−bkgd).

TABLE 3 Sum of Tables 1 & 2 specific activity- specific activity-adjusted, mean copies adjusted, mean RLU − bkgd, input RLU − bkgd x k =46.6  0 10² 47,388 47,388 10³ 448,100 448,100 10⁴ 3,160,250 11,812,85010⁵ 34,458,663 67,477,623 10⁶ 80,575,713 327,187,353 10⁷ 114,761,2381,694,928,918 10⁸ 381,316,755 14,028,047,015 10⁹ 1,519,281,48866,898,644,008

Each of the replicates for the specific activity-adjusted, meanbackground-subtracted values for each probe and for the summed probevalues is shown in FIG. 2. Chemiluminescence from AE-labeled probe ofSEQ ID NO:5 plus unlabeled probe of SEQ ID NO:5 demonstrated a linearincrease in RLU from about 10³ to 10⁵ copies per reaction of RNAtranscript input. Chemiluminescence from AE-labeled probe of SEQ ID NO:6plus unlabeled probe of SEQ ID NO:6 demonstrated a linear increase inRLU from about 10⁵ to 10⁹ copies per reaction of RNA transcript input.The slopes of responses from the two sets of probes were aboutcollinear. Their summed RLU values demonstrate two discontinuous linearincreases in RLU from about 10³ to 10⁵ copies per reaction and fromabout 10⁴ to 10⁹ copies per reaction. However, transforming the RLUvalues from AE-labeled probe of SEQ ID NO:6 plus unlabeled probe of SEQID NO:6 by a multiplying by a constant (k), in this case 46.6,demonstrated the continuous linear dynamic range of detection from thesetwo sets of probes from about 10³ to 10⁹ copies per reaction (Tables 2and 3, FIG. 3). The constant (k) is a visualization tool used to adjustthe slopes of both linear regression to a common x-intercept, but is notnecessary for the quantification of the two regions of amplicons. Theconstant (k) was calculated by dividing the mean RLU value from a firstprobe by the mean RLU value from a second probe taken at a target inputconcentration in which RLU values from both probes are part of the samelinear regression. The value varies depending on at least the primerconcentrations, probe specific activities, and amplificationefficiencies.

Example 2: Quantitative Detection of Amplicons with AE-HICS Probes Overa Dynamic Range in the Same Reaction Chamber

This example demonstrates an embodiment that provides quantitativedetection of amplicons over a dynamic range from 10⁶ to 10¹⁰ copies oftarget input in the same reaction chamber.

At the completion of TMA reactions, 100 μL volumes of HybridizationReagent were added, mixed and incubated at room temperature for 20minutes. TMA Separation Suspension Reagent (250 μL) was added, mixed andincubated at 60° C. for 10 minutes. The tubes containing the abovesolutions were placed on a magnetic separation rack at room temperaturefor 5 minutes, the liquid phase was removed, 100 μL of probes inhalf-strength Hybridization Reagent were added, and the resultingmixtures were incubated at 60° C. for 30 minutes and at room temperaturefor 5 minutes. The probes used were (a) 10 nM self-quenching AE-labeledHICS probe of SEQ ID NO:7, (b) 100 nM self-quenching, wavelength-shiftedAE-labeled wsHICS probe of SEQ ID NO:8), or (c) both probes at theconcentrations indicated in (a) and (b). The relative placement of theprobes on the distinguishable amplicons is shown diagrammatically inFIG. 4. Labeled probe of SEQ ID NO:7 detects both amplicons, whereas thelabeled probe of SEQ ID NO:8 detects only the longer amplicons.

At the completion of the hybridization reactions, 100 μL volumes ofHybridization Diluent were added to the hybridization reactions, and theresulting mixtures were incubated at 60° C. for 10 minutes. The tubescontaining the above solutions were placed on a magnetic separation rackat room temperature for 5 minutes, the liquid phase was removed, and 100μL volumes of Read Reagent were added and mixed.

Chemiluminescence from these reactions was initiated in a modifiedluminometer equipped with two high-count photomultiplier tube modules(PMT; 28 mm diameter, head-on, bialkali cathode; Hamamatsu Photonics,Hamamatsu City, Japan) on opposite sides of and directed towards alight-tight detection chamber fitted with injector tubing from tworeagent pumps. Filters (25.4 mm diameter; 430AF60 from Omega Optical(Brattleboro, Vt.) and OG550 from Newport Corp. (Franklin, Mass.)) werefitted between the PMTs and the detection chamber to allowdiscrimination of emissions from the two probes. Similar multiplewavelength luminometers are known in the art (e.g., U.S. Pat. No.5,447,687 and Clin. Chem. 29(9), 1604-1608 (1983)). Chemiluminescencefrom these reactions was initiated by addition of 200 μL DetectionReagent 3, a two second pause prior to addition of 200 μL DetectionReagent 4, then simultaneous acquisition of 100×0.4 second intervals oftime-resolved chemiluminescent data from both channels with no delaysbetween intervals.

The chemiluminescent output in RLUs from these intervals is reported inTables 4 and 5. The last column in each table reflects the sum of theindividual results.

TABLE 4 10 nM AE-labeled probe 10 nM AE-labeled 100 nM AE-labeled of SEQID NO: 7 probe of probe of 100 nM AE-labeled probe PMT1 SEQ ID NO: 7 SEQID NO: 8 of SEQ ID NO: 8 copies input rep RLU RLU - bkgd RLU RLU - bkgdRLU RLU - bkgd  0 1 377 1,176 1,487  0 2 390 1,117 1,498 10² 1 465 821,544 52 10² 2 622 239 2,161 669 10³ 1 526 143 1,868 376 10³ 2 669 2861,632 140 10⁴ 1 641 258 1,529 37 10⁴ 2 705 322 1,823 331 10⁵ 1 660 2771,405 259 1,631 139 10⁵ 2 836 453 1,165 19 1,783 291 10⁶ 1 1,128 7451,072 −75 1,974 482 10⁶ 2 1,389 1,006 1,154 8 2,160 668 10⁷ 1 2,9732,590 1,448 302 3,376 1,884 10⁷ 2 2,667 2,284 1,312 166 3,741 2,249 10⁸1 1,376 230 7,624 6,132 10⁸ 2 1,423 277 8,208 6,716 10⁹ 1 2,027 88111,281 9,789 10⁹ 2 2,343 1,197 13,897 12,405  10¹⁰ 1 5,496 4,350 19,73618,244  10¹⁰ 2 5,726 4,580 20,159 18,667

TABLE 5 10 nM AE-labeled probe of 10 nM AE-labeled 100 nM AE-labeled SEQID NO: 7 probe of probe of 100 nM AE-labeled probe PMT2 SEQ ID NO: 7 SEQID NO: 8 of SEQ ID NO: 8 copies input rep RLU RLU - bkgd RLU RLU - bkgdRLU RLU - bkgd  0 1 314 16,147 16,464  0 2 767 15,321 16,006 10² 1 63393 18,291 2,056 10² 2 642 102 26,609 10,374 10³ 1 614 74 19,620 3,38510³ 2 630 90 18,419 2,184 10⁴ 1 613 73 17,711 1,476 10⁴ 2 629 89 21,9875,752 10⁵ 1 642 102 21,097 5,363 17,883 1,648 10⁵ 2 629 89 16,355 62119,147 2,912 10⁶ 1 689 149 15,224 −510 14,504 −1731 10⁶ 2 642 102 17,2581,524 16,087 −148 10⁷ 1 702 162 24,409 8,675 20,506 4,271 10⁷ 2 719 17920,781 5,047 24,571 8,336 10⁸ 1 28,655 12,921 39,411 23,176 10⁸ 2 26,90011,166 49,451 33,216 10⁹ 1 70,691 54,957 92,458 76,223 10⁹ 2 80,23664,502 118,738 102,503  10¹⁰ 1 283,009 267,275 273,576 257,341  10¹⁰ 2308,126 292,392 309,765 293,530

The mean RLU from the zero RNA transcript input (first entry in eachtable) was used as the background (“bkgd”) measurement and wassubtracted from the results of experiments containing (a) 10², 10³, 10⁴,10⁵, 10⁶ and 10⁷ copies RNA transcripts input for AE-labeled probe ofSEQ ID NO:7, (b) 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ and 10¹⁰ copies RNA transcriptsinput for AE-labeled probe of SEQ ID NO:8, or (c) 10², 10³, 10⁴, 10⁵,10⁶, 10⁷, 10⁸, 10⁹ and 10¹⁰ copies RNA transcripts input for both probes(Tables 4 and 5, FIGS. 5 and 6).

FIG. 5 demonstrates that low wavelength RLU (captured by filtered PMT1)response from AE-labeled probe of SEQ ID NO:7 corresponds linearly fromat least about 10⁶ to 10⁷ copies per reaction of RNA transcript inputand that this response is very similar when the probe was used alone orin concert with AE-labeled probe of SEQ ID NO:8.

FIG. 6 demonstrates that high wavelength RLU (captured by filtered PMT2)response from AE-labeled probe of SEQ ID NO:8 corresponds linearly fromat least about 10⁷ to 10⁹ copies per reaction of RNA transcript inputand that this response is very similar whether the probe was used aloneor in concert with AE-labeled probe of SEQ ID NO:7.

FIG. 7 demonstrates that when each reaction is simultaneously treatedwith both probes, chemiluminescent data acquired from single tubes wasseparated into low and high wavelength emissions by filtered PMT1 andfiltered PMT2. FIG. 7 clearly demonstrates that RLU from these two setsof emissions in single tubes allows linear quantitative detection ofamplicons from about 10⁶ to 10¹⁰ copies per reaction of RNA transcriptinput.

Example 3: Quantitative Detection of Amplicons with AE-HICS Probes Overa Dynamic Range in the Same Reaction Chamber

This example demonstrates an embodiment that provides quantitativedetection of amplicons over a dynamic range from 10⁴ to 10¹⁰ copies oftarget input in the same reaction chamber.

At the completion of TMA reactions, 100 μL volumes of HybridizationReagent were added and mixed. Separation Reagent (50 μL) was added, andthe resulting mixtures were incubated at 60° C. for 10 minutes and atroom temperature for 10 minutes. The tubes containing the abovesolutions were placed on a magnetic separation rack at room temperaturefor 5 minutes, the liquid phase was removed, 100 μL of probes inHybridization Reagent were added, mixed and incubated at 60° C. for 15minutes and at room temperature for 5 minutes. The same probes,combinations of probes, and concentrations were used as in Example 2.

At the completion of the hybridization reactions, the tubes containingthe above solutions were placed on a magnetic separation rack at roomtemperature for 5 minutes and the liquid phase was removed. Next, 100 μLof Hybridization Reagent was added, mixed, placed on a magneticseparation rack at room temperature for 5 minutes and the liquid phasewas removed. Finally, 100 μL volumes of Read Reagent were added andmixed.

Chemiluminescence from these reactions was acquired as in Example 2. Thechemiluminescent output in RLUs from these intervals is reported inTables 6 and 7. The last column in each table reflects the sum of theindividual results.

TABLE 6 10 nM AE-labeled probe of 10 nM AE-labeled 100 nM AE-labeled SEQID NO: 7 probe of probe of 100 nM AE-labeled probe PMT1 SEQ ID NO: 7 SEQID NO: 8 of SEQ ID NO: 8 copies input rep RLU RLU - bkgd RLU RLU - bkgdRLU RLU - bkgd  0 1 241 2,186 2,083  0 2 274 2,205 2,044 10² 1 265 82,005 −59 10² 2 285 28 2,108 45 10³ 1 282 25 2,091 28 10³ 2 308 51 2,07512 10⁴ 1 345 88 2,289 226 10⁴ 2 350 93 2,042 −22 10⁵ 1 949 692 2,103 −932,600 537 10⁵ 2 877 620 2,027 −169 2,594 531 10⁶ 1 2,481 2,224 2,138 −584,386 2,323 10⁶ 2 2,685 2,428 2,066 −130 4,514 2,451 10⁷ 1 6,093 5,8362,458 263 8,162 6,099 10⁷ 2 6,044 5,787 2,347 152 7,431 5,368 10⁸ 13,570 1,375 12,833 10,770 10⁸ 2 3,188 993 13,244 11,181 10⁹ 1 7,2385,043 22,468 20,405 10⁹ 2 6,904 4,709 21,679 19,616  10¹⁰ 1 18,43916,244 35,697 33,634  10¹⁰ 2 18,364 16,169 37,785 35,722

TABLE 7 10 nM AE-labeled probe of 10 nM AE-labeled 100 nM AE-labeled SEQID NO: 7 probe of probe of 100 nM AE-labeled probe PMT2 SEQ ID NO: 7 SEQID NO: 8 of SEQ ID NO: 8 copies input rep RLU RLU - bkgd RLU RLU - bkgdRLU RLU - bkgd  0 1 408 49,859 47,502  0 2 443 49,554 44,108 10² 1 46540 44,440 −1365 10² 2 456 31 45,037 −768 10³ 1 471 46 44,644 −1161 10³ 2458 33 45,240 −565 10⁴ 1 492 67 48,723 2,918 10⁴ 2 509 84 41,912 −389310⁵ 1 479 54 50,653 947 49,501 3,696 10⁵ 2 527 102 48,818 −889 44,034−1771 10⁶ 1 551 126 50,255 549 51,810 6,005 10⁶ 2 566 141 48,679 −102851,876 6,071 10⁷ 1 645 220 64,907 15,201 67,800 21,995 10⁷ 2 673 24859,778 10,072 61,545 15,740 10⁸ 1 143,945 94,239 114,205 68,400 10⁸ 2113,554 63,848 127,507 81,702 10⁹ 1 393,948 344,242 383,192 337,387 10⁹2 351,286 301,580 376,625 330,820  10¹⁰ 1 1,029,786 980,080 997,605951,800  10¹⁰ 2 1,034,798 985,092 1,011,646 965,841

The mean RLU from the zero RNA transcript input (first entry in eachtable) was used as the background (“bkgd”) measurement and wassubtracted from the results of experiments containing (a) 10², 10³, 10⁴,10⁵, 10⁶, and 10⁷ copies RNA transcripts input for AE-labeled probe ofSEQ ID NO:7, (b) 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ and 10¹⁰ copies RNA transcriptsfor AE-labeled probe of SEQ ID NO:8, or (c) 10², 10³, 10⁴, 10⁵, 10⁶,10⁷, 10⁸, 10⁹ and 10¹⁰ copies RNA transcripts for both probes (Tables 6and 7, FIGS. 8 and 9).

FIG. 8 demonstrates that low wavelength RLU (captured by filtered PMT1)response from AE-labeled probe of SEQ ID NO:7 corresponds linearly fromat least about 10⁴ to 10⁶ copies per reaction of RNA transcript inputand that this response is similar whether the probe is used alone or inconcert with AE-labeled probe of SEQ ID NO:8.

FIG. 9 demonstrates that high wavelength RLU (captured by filtered PMT2)response from AE-labeled probe of SEQ ID NO:8) corresponds linearly fromat least about 10⁷ to 10¹⁰ copies per reaction of RNA transcript inputand that this response is very similar whether the probe is used aloneor in concert with AE-labeled probe of SEQ ID NO:7.

FIG. 10 demonstrates that when each reaction is simultaneously treatedwith both probes, chemiluminescent data acquired from single tubes wasseparated into low and high wavelength emissions by filtered PMT1 andfiltered PMT2. FIG. 10 clearly demonstrates that RLU from these two setsof emissions in single tubes allows linear quantitative detection ofamplicons from at least about 10⁴ to 10¹⁰ copies per reaction of RNAtranscript input.

Example 4: Quantitative Detection of Amplicons with AE-FluorophoreProbes Over a Dynamic Range in the Same Reaction Chamber

This example describes steps for detection of amplified products over abroad dynamic range of input target nucleic acids in the same reactionchamber.

TMA reactions are performed as described in the “Amplification Methods”section above. Hybridization workup and probe selection is performed asin Examples 2 or 3. The probes are present in total concentrationssimilar to those in the previous examples, i.e., sufficient to span theranges of the amplicons synthesized and the detection apparatus. One ofthe probes has a standard type of AE attached to an oligonucleotide, asin Example 1. The other probe has a conjugate of an AE linked to afluorophore, similar to AE conjugates with rhodamine and Texas Reddescribed in U.S. Pat. No. 6,165,800 (emission spectra in FIGS. 1B and1C), with the AE-fluorophore moiety attached to an oligonucleotide.

The luminescent probes are distinguishable from one another by differentwavelength ranges of electromagnetic radiation emitted by each probeafter initiation by addition and mixing of appropriate chemicals similarto those described in the previous examples, especially Example 1.Output emissions from a single detection vessel containing both thelonger and shorter amplicons are simultaneously collected and separatedinto their respective wavelength ranges with, for example, a multiplewavelength luminometer like the one described in Example 2. The outputfrom the first wavelength range corresponds to the first probehybridized to both amplicons; the output from the second wavelengthrange corresponds to the second probe hybridized only to one of theamplicons. Alternately, the output from the second wavelength rangecorresponds to the first probe hybridized to both amplicons; the outputfrom the first wavelength range corresponds to the second probehybridized only to one of the amplicons. Unhybridized probes yield lowemissions due to designs in the previous examples, especially due totreatment with Alkaline Reagent as in Example 1.

The mean RLU from the zero RNA transcript input (background or bkgd) issubtracted from the results of experiments containing 10², 10³, 10⁴,10⁵, 10⁶, 10², 10⁸ and 10⁹ copies RNA transcripts input.Background-subtracted chemiluminescence from the first probedemonstrates a linear increase in RLU from about 10² to about 10⁶ copiesper reaction of RNA transcript input; background-subtractedchemiluminescence from the second probe demonstrates a linear increasein RLU from about 10⁵ to about 10⁹ copies per reaction of RNA transcriptinput. Alternately, background-subtracted chemiluminescence from thesecond probe demonstrates a linear increase in RLU from about 10² toabout 10⁶ copies per reaction of RNA transcript input;background-subtracted chemiluminescence from the first probedemonstrates a linear increase in RLU from about 10⁵ to about 10⁹ copiesper reaction of RNA transcript input. The slopes of responses from thetwo sets of probes are co-linear. Linear output responses from these twoprobes overlap. However, taken together, the output from these twoprobes demonstrates quantitative detection of amplicons from about 10²to about 10⁹ copies per reaction of RNA transcript input.

Example 5: Quantitative Detection of Amplicons with AE-HICS Probes Overa Dynamic Range in the Same Reaction Chamber

This example demonstrates methods that show quantitative detection ofamplified nucleic acid sequences resulting from target nucleic acidsover a dynamic range from about 10² to 10⁹ copies of input targetnucleic acid in the same reaction chamber.

At the completion of TMA reactions, samples were processed using methodssubstantially similar to those described in Examples 2 and 3. The probesused were (a) 10 nM self-quenching AE-labeled probe of SEQ ID NO:7, (b)100 nM self-quenching, wavelength-shifted AE-labeled probe of SEQ IDNO:8, or (c) both probes at the concentrations indicated for individualprobes in (a) and (b). The relative placement of the probes on thedistinguishable amplicons is shown diagrammatically in FIG. 4. TheAE-labeled probe of SEQ ID NO:7 is able to detect both amplicons,whereas the AE-labeled probe of SEQ ID NO:8 is only able to detect oneof the amplicons.

Chemiluminescence from these reactions was acquired as in Examples 2 and3 except the probes were pretreated and then the detection reactionswere initiated by addition of 200 μL of Detection Reagent 5 instead ofDetection Reagent 3 followed by addition of 200 μL Detection Reagent 4.The chemiluminescent output in RLUs from these intervals is reported inTables 8 and 9. The last column in each table reflects the sum of theindividual probe outputs.

TABLE 8 10 nM AE-labeled probe of 10 nM AE-labeled 100 nM AE-labeled SEQID NO: 7 probe of probe 100 nM AE-labeled probe PMT1 SEQ ID NO: 7 of SEQID NO: 8 of SEQ ID NO: 8 copies input rep RLU RLU - bkgd RLU RLU - bkgdRLU RLU - bkgd  0 1 14,562 26,106 36,423  0 2 14,438 24,472 36,827  0 314,082 26,331 36,803 10² 1 14,794 433 25,179 −457 35,358 −1326 10² 214,929 568 27,807 2,171 37,238 554 10² 3 14,862 501 27,130 1,494 37,488804 10³ 1 15,678 1,317 25,340 −296 39,793 3,109 10³ 2 21,002 6,64127,005 1,369 38,311 1,627 10³ 3 16,434 2,073 26,027 391 38,101 1,417 10⁴1 24,766 10,405 25,362 −274 51,125 14,441 10⁴ 2 23,483 9,122 25,919 28347,760 11,076 10⁴ 3 26,572 12,211 25,078 −558 54,378 17,694 10⁵ 1 72,55958,198 26,854 1,218 121,786 85,102 10⁵ 2 78,041 63,680 25,406 −230106,645 69,961 10⁵ 3 95,444 81,083 23,635 −2001 115,123 78,439 10⁶ 1186,102 171,741 25,108 −528 203,978 167,294 10⁶ 2 191,297 176,936 26,075439 271,260 234,576 10⁶ 3 199,336 184,975 24,664 −972 255,668 218,98410⁷ 1 588,202 573,841 27,084 1,448 586,704 550,020 10⁷ 2 413,767 399,40626,679 1,043 602,568 565,884 10⁷ 3 450,198 435,837 28,763 3,127 526,836490,152 10⁸ 1 723,259 708,898 37,317 11,681 961,790 925,106 10⁸ 2723,269 708,908 39,453 13,817 944,710 908,026 10⁸ 3 763,899 749,53837,161 11,525 984,831 948,147 10⁹ 1 1,034,534 1,020,173 55,336 29,7001,100,567 1,063,883 10⁹ 2 994,327 979,966 54,090 28,454 1,136,9501,100,266 10⁹ 3 1,076,328 1,061,967 52,833 27,197 1,130,972 1,094,288 10¹⁰ 1 1,110,316 1,095,955 60,085 34,449 1,106,720 1,070,036  10¹⁰ 21,080,085 1,065,724 59,140 33,504 1,149,267 1,112,583  10¹⁰ 3 1,136,1361,121,775 56,674 31,038 1,112,549 1,075,865

TABLE 9 10 nM AE-labeled probe of 10 nM AE-labeled 100 nM AE-labeled SEQID NO: 7 probe of probe of 100 nM AE-labeled probe PMT2 SEQ ID NO: 7 SEQID NO: 8 of SEQ ID NO: 8 copies input rep RLU RLU - bkgd RLU RLU - bkgdRLU RLU - bkgd  0 1 2,952 236,010 208,897  0 2 2,913 232,904 221,790  03 3,112 227,080 233,341 10² 1 3,013 21 196,451 −35547 206,041 −15302 10²2 3,063 71 315,433 83,435 203,440 −17903 10² 3 3,159 167 223,865 −8133194,955 −26388 10³ 1 3,126 134 200,219 −31779 238,950 17,607 10³ 2 3,231239 218,126 −13872 201,506 −19837 10³ 3 3,165 173 216,653 −15345 210,847−10496 10⁴ 1 3,335 343 242,834 10,836 254,037 32,694 10⁴ 2 3,388 396230,274 −1724 189,268 −32075 10⁴ 3 3,507 515 231,765 −233 299,412 78,06910⁵ 1 4,250 1,258 262,302 30,304 199,632 −21711 10⁵ 2 4,139 1,147262,013 30,015 211,301 −10042 10⁵ 3 4,527 1,535 203,150 −28848 220,057−1286 10⁶ 1 5,777 2,785 250,081 18,083 224,196 2,853 10⁶ 2 5,973 2,981257,312 25,314 229,233 7,890 10⁶ 3 6,076 3,084 246,642 14,644 227,1065,763 10⁷ 1 12,273 9,281 453,599 221,601 461,451 240,108 10⁷ 2 9,7046,712 373,223 141,225 646,791 425,448 10⁷ 3 10,029 7,037 521,274 289,276332,741 111,398 10⁸ 1 14,422 11,430 1,326,590 1,094,592 1,280,8701,059,527 10⁸ 2 14,559 11,567 1,466,518 1,234,520 1,238,109 1,016,76610⁸ 3 15,583 12,591 1,368,674 1,136,676 1,283,745 1,062,402 10⁹ 1 19,04716,055 4,248,797 4,016,799 4,424,877 4,203,534 10⁹ 2 19,191 16,1994,357,020 4,125,022 4,280,862 4,059,519 10⁹ 3 20,178 17,186 4,228,4403,996,442 3,605,149 3,383,806  10¹⁰ 1 20,793 17,801 6,357,492 6,125,4946,096,242 5,874,899  10¹⁰ 2 21,125 18,133 6,265,950 6,033,952 6,208,7765,987,433  10¹⁰ 3 20,856 17,864 6,316,084 6,084,086 5,881,110 5,659,767

The mean RLU from the zero RNA transcript input (first entry in eachtable) was used as the background (“bkgd”) measurement and wassubtracted from the results of experiments containing 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹ and 10¹⁶ copies RNA transcripts input forAE-labeled probes of SEQ ID NO:7, SEQ ID NO:8, or both probes (Tables 8and 9, FIGS. 11 and 12).

FIG. 11 demonstrates that low wavelength RLU (captured by filtered PMT1)response from AE-labeled probe of SEQ ID NO:7 corresponds linearly fromat least about 10² to 10⁷ copies per reaction of RNA transcript inputand that this response is similar whether the probe is used alone or inconcert with AE-labeled probe of SEQ ID NO:8.

FIG. 12 demonstrates that high wavelength RLU (captured by filteredPMT2) response from AE-labeled probe of SEQ ID NO:8 corresponds linearlyfrom at least about 10⁷ to 10⁹ copies per reaction of RNA transcriptinput and that this response is very similar whether the probe is usedalone or in concert with AE-labeled probe of SEQ ID NO:7.

FIG. 13 demonstrates that when each reaction is simultaneously treatedwith both probes, chemiluminescent data acquired from single tubes wasseparated into low and high wavelength emissions by filtered PMT1 andfiltered PMT2. FIG. 13 clearly demonstrates that RLU from these two setsof emissions in single tubes allows linear quantitative detection ofamplicons from about 10² to 10⁹ copies per reaction of RNA transcriptinput.

Example 6: Detection of Distinguishable Amplicons Made Using Modifiedand Unmodified Primers

This example demonstrates quantitative detection of amplicons from overa range of 10³ to 10⁹ copies of target nucleic acid in the same reactiontubes. The amplification oligonucleotides are a first promoter providerand a second promoter provider wherein the two promoter providerscomprise substantially identical nucleotide sequences except that atleast one of the two promoter providers is modified so to produce adistinguishable amplicon relative to that produced by the other promoterprovider. The first and the second promoter providers are also presentin the TMA reaction in different concentrations so as to generate adifferentiable ratio of their respective amplicons.

Using the illustrative amplification system described above, the firstpromoter provider is SEQ ID NO:1 and the second promoter provider issubstantially identical to SEQ ID NO:1 except that the targethybridizing sequence contains a modified nucleobase. The first promoterprovider is present in the amplification reaction at a concentrationthat is 1000 times in excess as the concentration for the secondpromoter provider. In addition, SEQ ID NO:2 is used as the amplificationoligonucleotide member in conjunction with both the first and the secondpromoter providers. Isothermal amplification is performed.

At the completion of the TMA amplification reactions, 100 μL volumes ofprobes in Hybridization Reagent is added to the amplification reactionmixtures, which are mixed and incubated at 60° C. for 15 minutes, whichwill allow hybridization of probes specific for complementary sequencesin the synthesized amplicons. The probes are: (a) an AE-labeled probethat hybridizes to the amplicon produced using the first promoterprovider but not the amplicon produced using the second promoterprovider; and (b) AE-labeled probe that hybridizes to the ampliconproduced using the second promoter provider but not the ampliconproduced using the first promoter provider. These AE-labeled probeshybridize their respective amplicons at a position that includes thedistinguishable position represented by the modification present in thesecond promoter provider but not the first promoter provider.

Chemiluminescence from these reactions is acquired essentially as inExample 5. Reactions can be initiated by addition of 200 μL DetectionReagent 5 instead of Detection Reagent 3.

Example 7: Detection of Distinguishable Products Made UsingPromoter-Based Amplification Oligomers with 5′ Unpaired Base(s) ProvidedBetween the 3′ End of their Promoter Sequences Ans the 5′ End of theirTarget Hybridizing Sequences

This example demonstrates quantitative detection of amplicons over arange of 10³ to 10⁹ copies of target nucleic acid in the same reactiontubes. The amplification oligonucleotides are a first promoteramplification oligonucleotide and a second promoter oligonucleotide,wherein the two promoter amplification oligonucleotides comprisesubstantially identical nucleotide sequences except that each of the twopromoter amplification oligonucleotides comprise between the 5′ end oftheir target hybridizing sequences and the 3′ end of their promotersequences, at least one separately unique unpaired base so to produce adistinguishable amplicon relative to that produced by the other promoteramplification oligonucleotide. The first and the second promoteroligonucleotides are also present in the TMA reaction in differentconcentrations so as to generate a differentiable ratio of theirrespective amplicons.

Using the illustrative amplification system described above, the firstpromoter oligonucleotide is SEQ ID NO:1 further comprising a 5′ unpairedbase (5′-AATTTAATACGACTCACTATAGGGAGA(X) GTTTGTATGTCTGTTGCTATTAT-3′ (5′unpaired base X version of SEQ ID NO:1, wherein X represents one or moreunpaired bases)) and the second promoter oligonucleotide is also SEQ IDNO:1 further comprising a 5′ unpaired base that is distinguishable fromthat present on the first promoter oligonucleotide(5′-AATTTAATACGACTCACTATAGGGAGA(Y)GTTTGTATGTCTGTTGCTATTAT-3′ (5′unpaired base Y version of SEQ ID NO:1, wherein Y represents one or moreunpaired bases and wherein the contiguous nucleotide sequence Y is notthe same as the contiguous nucleotide sequence X)). The first promoteroligonucleotide is present in the amplification reaction at aconcentration that is 1000 times in excess as the concentration for thesecond promoter oligonucleotide. In addition, SEQ ID NO:2 is used as theamplification oligonucleotide member in conjunction with both the firstand the second promoter oligonucleotides. Isothermal amplification isperformed.

At the completion of the TMA amplification reactions, 100 μL volumes ofprobes in Hybridization Reagent are added to the amplification reactionmixtures, which are mixed and incubated at 60° C. for 15 minutes toallow hybridization of probes specific for complementary sequences inthe synthesized amplicons. The probes are: (a) an AE-labeled probe thathybridizes to the amplicon produced using the first promoteroligonucleotide but not the amplicon produced using the second promoteroligonucleotide; and (b) AE-labeled probe that hybridizes to theamplicon produced using the second promoter oligonucleotide but not theamplicon produced using the first promoter oligonucleotide. TheseAE-labeled probes hybridize their respective amplicons at a positionthat includes the distinguishable position represented by themodification present in the second promoter oligonucleotide but not thefirst promoter oligonucleotide.

Chemiluminescence from these reactions is acquired essentially as inExample 5.

Example 8: Detection of Distinguishable Products Made UsingAmplification Oligonucleotides with 5′ Unpaired Bases

This example demonstrates quantitative detection of amplicons over arange of 10³ to 10⁹ copies of target nucleic acid in the same reactiontubes. The amplification oligonucleotides are a first amplificationoligonucleotide and a second oligonucleotide wherein the twoamplification oligonucleotides comprise substantially identicalnucleotide sequences except that each of the two amplificationoligonucleotides comprise a separately unique 5′ unpaired base so toproduce a distinguishable amplicon relative to that produced by theother amplification oligonucleotide. The first and the secondamplification oligonucleotides are also present in the TMA reaction indifferent concentrations so as to generate a differentiable ratio oftheir respective amplicons.

Using the illustrative amplification system described above, the firstamplification oligonucleotide is SEQ ID NO:2 further comprising a 5′unpaired base (5′-(X)ACAGCAGTACAAATGGCAG-3′ (5′ unpaired base X versionof SEQ ID NO:2, wherein X represents one or more unpaired bases)) andthe second amplification oligonucleotide is also SEQ ID NO:2 furthercomprising a 5′ unpaired base that is distinguishable from that presenton the first promoter oligonucleotide (5′-(Y)ACAGCAGTACAAATGGCAG-3′ (5′unpaired base Y version of SEQ ID NO:2, wherein Y represents one or moreunpaired bases and wherein the contiguous nucleotide sequence Y is notthe same as the contiguous nucleotide sequence X)). In this example, Xis a contiguous nucleic acid sequence that is 5 nucleobases in lengthand Y is a contiguous nucleic acid sequence that is 5 nucleobases inlength, wherein the contiguous nucleic acid sequences of X and Y aredissimilar thus allowing for separately detecting and distinguishingeach subsequent amplicon species. The first amplificationoligonucleotide is present in the amplification reaction at aconcentration that is 1000 times in excess as the concentration for thesecond amplification oligonucleotide. In addition, SEQ ID NO:1 is usedas the promoter amplification oligonucleotide member in conjunction withboth the first and the second amplification oligonucleotides. Isothermalamplification is performed.

At the completion of the TMA amplification reactions, 100 μL volumes ofprobes in Hybridization Reagent are added to the amplification reactionmixtures, which are mixed and incubated at 60° C. for 15 minutes, whichallows hybridization of probes specific for complementary sequences inthe synthesized amplicons. The probes are: (a) an AE-labeled probe thathybridizes to the amplicon produced using the first amplificationoligonucleotide but not the amplicon produced using the secondamplification oligonucleotide; and (b) AE-labeled probe that hybridizesto the amplicon produced using the second amplification oligonucleotidebut not the amplicon produced using the first amplificationoligonucleotide. These AE-labeled probes hybridize their respectiveamplicons at a position that includes the distinguishable positionrepresented by the modification present in the second promoteramplification oligonucleotide but not the first promoter amplificationoligonucleotide.

Chemiluminescence from these reactions is acquired essentially as inExample 5.

Example 9: Detection of Distinguishable Products Made Using PromoterAmplification Oligonucleotides with Different Promoter Sequences

This example demonstrates quantitative detection of amplicons over arange of 10³ to 10⁹ copies of target nucleic acid in the same reactiontubes. The amplification oligonucleotides are a first promoter-basedamplification oligonucleotide and a second promoter-based amplificationoligonucleotide wherein the two promoter-based amplificationoligonucleotides comprise substantially identical nucleotide sequencesexcept that at least one of the two promoter-based amplificationoligonucleotides is modified so to produce a distinguishable ampliconrelative to that produced by the other promoter-based amplificationoligonucleotide (for example, as in Example 6). The first and the secondpromoter-based amplification oligonucleotides are present in the TMAreaction in similar concentrations. The promoter sequence of the secondpromoter-based amplification oligonucleotide is modified relative to thepromoter sequence of the first promoter-based amplificationoligonucleotide so as to generate a differentiable ratio of theirrespective amplicons.

Using the illustrative amplification system described above, the firstpromoter-based amplification oligonucleotide is SEQ ID NO:1 and thesecond promoter-based amplification oligonucleotide is substantiallyidentical to SEQ ID NO:1 except that the target hybridizing sequencecontains a modified nucleobase. In addition, the second promoter-basedamplification oligonucleotide contains a change to its promoter sequence(e.g., a modified nucleobase, missing nucleobase, extra nucleobase ornon-deoxyribonucleotide nucleobase, e.g., a ribonucleotide nucleobase).The promoter sequence of the second promoter-based amplificationoligonucleotide is about 1000 times less efficient in synthesizing RNAtranscripts than the unmodified promoter sequence of the firstpromoter-based amplification oligonucleotide. In addition, SEQ ID NO:2is used as the amplification oligonucleotide member in conjunction withboth the first and the second promoter-based amplificationoligonucleotides. Isothermal amplification is performed.

At the completion of the TMA amplification reactions, 100 μL volumes ofprobes in Hybridization Reagent are added to the amplification reactionmixtures, which are mixed and incubated at 60° C. for 15 minutes, whichallows hybridization of probes specific for complementary sequences inthe synthesized amplicons. The probes are: (a) an AE-labeled probe thathybridizes to the amplicon produced using the first promoter-basedamplification oligonucleotide but not the amplicon produced using thesecond promoter-based amplification oligonucleotide; and (b) AE-labeledprobe that hybridizes to the amplicon produced using the secondpromoter-based amplification oligonucleotide but not the ampliconproduced using the first promoter-based amplification oligonucleotide.These AE-labeled probes hybridize their respective amplicons at aposition that includes the distinguishable position represented by themodification present in the second promoter-based amplificationoligonucleotide but not the first promoter-based amplificationoligonucleotide.

Chemiluminescence from these reactions is acquired essentially as inExample 5.

Example 10: Distinguishable DNA and RNA Products

This example demonstrates quantitative detection of amplicons over arange of 10³ to 10⁹ copies of target nucleic acid in the same reactiontubes. The amplification oligonucleotides used herein are a singleprimer and a single promoter provider. The distinguishable amplificationproducts are (i) the DNA amplicons made during some of the steps of theTMA reaction, and (ii) the RNA transcripts made during some of the stepsof the TMA reaction. DNA and RNA are generated in TMA such that the RNAamplification products are in excess compared to the DNA amplificationproducts so as to generate a differentiable ratio of these respectiveamplicons.

Using the illustrative amplification system described above, theamplification oligonucleotide is SEQ ID NO:2, and SEQ ID NO:1 is used asthe promoter amplification oligonucleotide member. Isothermalamplification is performed.

At the completion of the TMA amplification reactions, 100 μL volumes ofprobes in Hybridization Reagent are added to the amplification reactionmixtures, which are mixed and incubated at 60° C. for 15 minutes, whichallows hybridization of probes specific for complementary sequences inthe synthesized amplicons. The probes are: (a) an AE-labeled probe thathybridizes to the DNA amplicon; and (b) an AE-labeled probe thathybridizes to the RNA amplicon. The probe hybridizing to the DNAamplicon preferably hybridizes to the strand that is antisense to theRNA amplicon. These AE-labeled probes hybridize their respectiveamplicons at positions that are distinguishable.

Chemiluminescence from these reactions is acquired essentially as inExample 5.

Example 11: Detection of Distinguishable Products Made UsingDistinguishable Nucleotide Triphosphates (NTPs)

This example demonstrates quantitative detection of amplicons over arange of 10³ to 10⁹ copies of target nucleic acid in the same reactiontubes. The amplification oligonucleotides used herein are a singleamplification oligonucleotide and a single promoter amplificationoligonucleotide. A fifth NTP is included in the amplificationcomposition. The NTP mix, then, is A, T, G and a ratio of C and IsoC,wherein the amount of C is greater than the amount of IsoC. Ampliconsspecies incorporate the C of the IsoC at an unequal ratio thatapproximately equals the unequal ratio of C:IsoC provided into theamplification reaction. Therefore, the probe binding site on eachamplicon species has either a C or an IsoC, which providesdifferentiable amplicons species at unequal amounts (e.g., (i) the RNAamplicon species having A, C, T and G incorporated into the probebinding site, and (ii) the RNA amplicon species having A, IsoC, T and Gincorporated into the probe binding site).

Using the illustrative amplification system described above, theamplification oligonucleotide is SEQ ID NO:2, and SEQ ID NO:1 is used asthe promoter amplification oligonucleotide member. The NTP mix is A, T,G, and C:IsoC in an 1000:1 ratio. Isothermal amplification is performed.

At the completion of the TMA amplification reactions, 100 μL volumes ofprobes in Hybridization Reagent are added to the amplification reactionmixtures, which are mixed and incubated at 60° C. for 15 minutes, whichallows hybridization of probes specific for complementary sequences inthe synthesized amplicons. The probes are: (a) an AE-labeled probe thatis 12-nt in length and comprises a contiguous nucleotide sequence thatis made of the natural NTPs so that the probe hybridizes and detects themore abundant amplicon species that incorporate the native C residueinto their primer binding site; and (b) an AE-labeled probe that is12-nt in length and comprises a contiguous nucleotide sequence that ismade of the natural A, T and C residues and IsoG residues so that theprobe hybridizes and detects the less abundant amplicon species thatincorporate the IsoC residue into their primer binding site. TheseAE-labeled probes hybridize their respective amplicons at positions thatare distinguishable.

Chemiluminescence from these reactions is acquired essentially as inExample 5.

Example 12: Detection of Distinguishable Products Made Using Two Pairsof Amplification Oligonucleotides that Synthesize Amplicons that do notOverlap

This example demonstrates quantitative detection of amplicons over arange of 10³ to 10⁹ copies of target nucleic acid in the same reactiontubes. The amplification oligonucleotides are a first set comprising afirst amplification oligonucleotide and a first promoter amplificationoligonucleotide, and a second set comprising a second amplificationoligonucleotide and a second promoter amplification oligonucleotide,wherein the two sets of amplification oligonucleotides comprisesubstantially non-overlapping target binding sites such that each of thetwo sets of amplification oligonucleotides produce a distinguishableamplicon relative to that produced by the other amplificationoligonucleotide. The first and the second sets of amplificationoligonucleotides are also present in the TMA reaction in differentconcentrations so as to generate a differentiable ratio of theirrespective amplicons.

Using the illustrative amplification system described above, the firstamplification oligonucleotide is SEQ ID NO:2 and the first promoteramplification oligonucleotide is SEQ ID NO:1, and the secondamplification oligonucleotide is SEQ ID NO:3 and the second promoteramplification oligomer has a target binding sequence that iscomplementary to the target sequence between SEQ ID NO:2 and SEQ ID NO:3and that is near the target sequence complementary to SEQ ID NO:2. Thefirst amplification oligonucleotide is present in the amplificationreaction at a concentration that is 1000 times in excess as theconcentration for the second amplification oligonucleotide. Isothermalamplification is performed.

At the completion of the TMA amplification reactions, 100 μL volumes ofprobes in Hybridization Reagent are added to the amplification reactionmixtures, which are mixed and incubated at 60° C. for 15 minutes, whichallows hybridization of probes specific for complementary sequences inthe synthesized amplicons. The probes are: (a) an AE-labeled probe thathybridizes to the amplicon produced using the first set of amplificationoligonucleotides but not the amplicon produced using the second set ofamplification oligonucleotides; and (b) AE-labeled probe that hybridizesto the amplicon produced using the second set of amplificationoligonucleotides but not the amplicon produced using the first set ofamplification oligonucleotides. These AE-labeled probes hybridize totheir respective amplicons at a position that includes thedistinguishable position represented by the unique sequences synthesizedbetween the first set of amplification oligonucleotides or between thesecond set of amplification oligonucleotides.

Chemiluminescence from these reactions is acquired essentially as inExample 5.

TABLE 10 SEQ ID NO: Sequence (5′ to 3′) Comment  1AATTTAATACGACTCACTATAGGGAGAGTTTGTATGTCTGTTGCTATTAT Promoter-primer  2ACAGCAGTACAAATGGCAG Primer  3 ATTCCCTACAATCCCCAAAGTCAA Primer  4GGGAGACAAGCUUGCAUGCCUGCAGGUCGACUCUAGAGGAUCCCCGGGUACCAGC in vitroACACAAAGGAAUUGGAGGAAAUGAACAAGUAGAUAAAUUAGUCAGUGCUGGAAUC transcriptAGGAAAAUACUAUUUUUAGAUGGAAUAGAUAAGGCCCAAGAUGAACAUGAGAAAUAUCACAGUAAUUGGAGAGCAAUGGCUAGUGAUUUUAACCUGCCACCUGUAGUAGCAAAAGAAAUAGUAGCCAGCUGUGAUAAAUGUCAGCUAAAAGGAGAAGCCAUGCAUGGACAAGUAGACUGUAGUCCAGGAAUAUGGCAACUAGAUUGUACACAUUUAGAAGGAAAAGUUAUCCUGGUAGCAGUUCAUGUAGCCAGUGGAUAUAUAGAAGCAGAAGUUAUUCCAGCAGAAACAGGGCAGGAAACAGCAUAUUUUCUUUUAAAAUUAGCAGGAAGAUGGCCAGUAAAAACAAUACAUACAGACAAUGGCAGCAAUUUCACCAGUGCUACGGUUAAGGCCGCCUGUUGGUGGGCGGGAAUCAAGCAGGAAUUUGGAAUUCCCUA  CAAUCCCCAAAGUCAAGGAGUAGUAGAAUCUAUGAAUAAAGAAUUAAAGAAAAUUAUAGGACAGGUAAGAGAUCAGGCUGAACAUCUUAAGACAGCAGUACAAAUGGCAGUAUUCAUCCACAAUUUUAAAAGAAAAGGGGGGAUUGGGGGGUACAGUGCAGGGGAAAGAAUAGUAGACAUAAUAGCAACAGACAUACAAACUAAAGAAUUACAAAAACAAAUUACAAAAAUUCAAAAUUUUCGGGUUUAUUACAGGGACAGCAGAAAUCCACUUUGGAAAGGACCAGCAAAGCUCCUCUGGAAAGGUGAAGGGGCAGUAGUAAUACAAGAUAAUAGUGACAUAAAAGUAGUGCCAAGAAGAAAAGCAAAGAUCAUUAGGGAUUAUGGAAAACAGAUGGCAGGUGAUGAUUGUGUGGCAAGUAGACAGGAUGAGGAUUAGAACAUGGAAAAGUUUAGUAAAACACCA  5 CCACAAUUUUAAAAGAAAAGGG Probe  6AGAAAAUUAUAGGACAGGUAAG Probe  7 CUCGUCCACAAUUUUAAAAGAAAAGGGACGAG Probe 8 CCUCUAGAAAAUUAUAGGACAGGUAAGAGAGG Probe  9GGGAGAGUUUGUAUGUCUGUUGCUAUUAUGUCUACUAUUCUUUCCCCUGCACUGU Amplicon  ACCCCCCAAUCCCCCCUUUUCUUUUAAAAUUGUGGAUGAAUACUGCCAUUUGUAC UGCUGU 10GGGAGAGUUUGUAUGUCUGUUGCUAUUAUGUCUACUAUUCUUUCCCCUGCACUGU AmpliconACCCCCCAAUCCCCCCUUUUCUUUUAAAAUUGUGGAUGAAUACUGCCAUUUGUACUGCUGUCUUAAGAUGUUCAGCCUGAUCUCUUACCUGUCCUAUAAUUUUCUUUAAUUCUUUAUUCAUAGAUUCUACUACUCCUUGACUUUGGGGAUUGUAGGGAAU

SEQ ID NO:1 is a sequence of a 50-nucleotide Promoter-primer. It may becomprised of one or more deoxyribonucleotides. Nucleotides 1-27 are onestrand of a T7 RNA polymerase promoter sequence, and nucleotides 27-50are complementary to a region of SEQ ID NO:4. SEQ ID NO:2 is a sequenceof a 19-nucleotide Primer. It may be comprised of one or moredeoxyribonucleotides. Nucleotides 1-19 are complementary to acomplementary sequence of a region of SEQ ID NO:4. SEQ ID NO:3 is asequence of a 24-nucleotide Primer. It may be comprised of one or moredeoxyribonucleotides. Nucleotides 1-24 are complementary to acomplementary sequence of a region of SEQ ID NO:4. SEQ ID NO:4 is asequence of a 1,016 nucleotide RNA in vitro transcript (IVT).Nucleotides 1-51 are from an RNA polymerase promoter sequence and acloning vector, and nucleotides 52-1,016 are from an HIV-1 genome thatincludes a 3′ region of the HIV-1 subtype B pol gene.

SEQ ID NO:5 is a sequence of a 22-nucleotide Probe. It may be comprisedof one or more 2′-O-methyl ribonucleotides. Nucleotides 1-22 arecomplementary to regions of SEQ ID NOS:9 and 10. It may have anattached, detectable label. The attached detectable label may be anacridinium ester. The acridinium ester may be of a composition toinclude a fluoro moiety at the ortho position of the phenyl ring asdisclosed in U.S. Pat. No. 5,840,873. The acridinium ester may beattached to a linker such as those derived from internucleotidyl linkerintermediates such as those described in U.S. Pat. No. 5,585,481. Theacridinium ester may be attached to the linker from the direction of theC9 position of the acridinium ester substantially as described in U.S.Pat. No. 5,185,439. The linker may be inserted between two nucleotidesof SEQ ID NO:5. The linker may be inserted between nucleotides 10 and 11of SEQ ID NO:5. SEQ ID NO:6 is a sequence of a 22-nucleotide Probe. Itmay be comprised of one or more 2′-O-methyl ribonucleotides. Nucleotides1-22 are complementary to a region of SEQ ID NO:10. It may have anattached, detectable label. The attached detectable label may be anacridinium ester. The acridinium ester may be of a composition toinclude a methyl moiety at the 2 position of the acridinium ring systemas disclosed in U.S. Pat. No. 5,840,873. The acridinium ester may beattached to a linker such as those derived from internucleotidyl linkerintermediates such as those described in U.S. Pat. No. 5,585,481. Theacridinium ester may be attached to the linker from the direction of theC9 position of the acridinium ester substantially as described in U.S.Pat. No. 5,185,439. The linker may be inserted between two nucleotidesof SEQ ID NO:6. The linker may be inserted between nucleotides 8 and 9of SEQ ID NO:6. SEQ ID NO:7 is a sequence of a 32-nucleotide Probe. Itmay be comprised of one or more 2′-O-methyl ribonucleotides. Nucleotides5-27 are complementary to regions of SEQ ID NOS:9 and 10, andnucleotides 1-7 and 26-32 are complementary to each other. It may havean attached, detectable label. The attached detectable label may be anacridinium ester. The acridinium ester may be of a composition asdisclosed in US Published Patent Appl. No. US 2007-0166759 A1. Theacridinium ester may be attached to a linker such as those derived frominternucleotidyl linker intermediates such as those described in U.S.Pat. No. 5,585,481 or from terminal linker intermediates such as5′-Amino-Modifier C6 (part number 10-1906-90) or 3′-Amino-Modifier C7CPG (part number 20-2958-01) from Glen Research Corporation, Sterling,Va. The acridinium ester may be attached to the linker from thedirection of the N10 position of the acridinium ester substantially asdescribed in US Published Patent Appl. No. US 2007-0166759 A1. Thelinker may be inserted between two nucleotides or at one of the terminiof SEQ ID NO:7. The linker may be at the 5′ terminus of SEQ ID NO:7. Theprobe may also have an attached quencher moiety that accepts the energyfrom the acridinium ester but does not reemit the energy as light. Thequencher may attached through an internucleotidyl or terminal linkeralternate to that used for the acridinium ester, through a linker on anucleotide base or as a dedicated phosphoramidite. The quencher may befrom the precursor 3′-BHQ-2 CPG (part number 20-5932-01) or 3′-DabcylCPG (part number 20-5912-01) from Glen Research Corporation, Sterling,Va. The quencher may be at the 3′ terminus of SEQ ID NO:7. SEQ ID NO:8is a sequence of a 32 nucleotide Probe. It may be comprised of one ormore 2′-O-methyl ribonucleotides. Nucleotides 6-30 are complementary toa region of SEQ ID NO:10, and nucleotides 1-5 and 28-32 arecomplementary to each other. It may have an attached, detectable label.The attached detectable label may be an acridinium ester. The acridiniumester may be of a composition as disclosed in US Published Patent Appl.No. US 2007-0166759 A1. The acridinium ester may be attached to a linkersuch as those derived from internucleotidyl linker intermediates such asthose described in U.S. Pat. No. 5,585,481 or from terminal linkerintermediates such as 5′-Amino-Modifier C6 (part number 10-1906-90) or3′-Amino-Modifier C7 CPG (part number 20-2958-01) from Glen ResearchCorporation, Sterling, Va. The acridinium ester may be attached to thelinker from the direction of the N10 position of the acridinium estersubstantially as described in US Published Patent Appl. No. US2007-0166759 A1. The linker may be inserted between two nucleotides orat one of the termini of SEQ ID NO:8. The linker may be at thepenultimate position to the 5′ terminus of SEQ ID NO:8. An energyacceptor moiety capable of accepting energy from the acridinium esterand reemission of a detectable form of energy (e.g., “light”),preferably of reemission of a detectable form of energy that can bedistinguished from the light emissions of the acridinium ester (e.g.,light of wavelengths different from those of the acridinium ester) maybe attached to the probe near the acridinium ester. The just describedenergy acceptor may be a fluorophore moiety. The just described energyacceptor may be at the 5′ terminus of SEQ ID NO:8. The just describedenergy acceptor may attached through an internucleotidyl or terminallinker alternate to that used for the acridinium ester, through a linkeron a nucleotide base or as a dedicated phosphoramidite. The fluorophoremay be a fluorescein or tetramethylrhodamine moiety. The probe may alsohave an attached quencher moiety that accepts the energy from theacridinium ester and/or from the energy acceptor attached near theacridinium ester but does not reemit the energy as light. The quenchermay attached through an internucleotidyl or terminal linker alternate tothose used for the acridinium ester or above described energy acceptor,through a linker on a nucleotide base or as a dedicated phosphoramidite.The quencher may be from the precursor 3′-BHQ-2 CPG (part number20-5932-01) or 3′-Dabcyl CPG (part number 20-5912-01) from Glen ResearchCorporation, Sterling, Va. The quencher may be at the 3′ terminus of SEQID NO:8.

SEQ ID NO:9 is a sequence of a 116-nucleotide amplicon. It may becomprised of one or more ribonucleotides. Nucleotides 1-5 are from anRNA polymerase promoter sequence, and nucleotides 6-116 arecomplementary to a region of SEQ ID NO:4. Probes of SEQ ID NOS: 5 and 7are at least partially complementary to a region of SEQ ID NO:9. SEQ IDNO:10 is a sequence of a 215-nucleotide amplicon. It may be comprised ofone or more ribonucleotides. Nucleotides 1-5 are from an RNA polymerasepromoter sequence, and nucleotides 6-215 are complementary to a regionof SEQ ID NO:4. The sequence of nucleotides 1-116 is shared with SEQ IDNO:9. Probes of SEQ ID NOS:5 and 7 are at least partially complementaryto a region of SEQ ID NO:10, and probes of SEQ ID NOS:6 and 8 are atleast partially complementary to a different region of SEQ ID NO:10.

All publications and patents mentioned in the above specification areherein incorporated by reference in their entirety for all purposeswhatsoever. Various modifications and variations of the described methodand system of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention that are obvious to those skilled in the relevant fields areintended to be within the scope of the following claims.

What is claimed is:
 1. A method of detecting a target nucleic acid in asample comprising the steps of: a. providing a sample suspected ofcontaining a target nucleic acid; b. generating from said target nucleicacid, a pre-defined ratio of at least two differentiable ampliconspecies, wherein said generating step is performed in a single vessel;and c. detecting the presence and amount of each generated ampliconspecies, wherein a first amplicon species is detectable in a firstlinear range representing a first concentration of target nucleic acidin said sample to a second concentration of target nucleic acid in saidsample and a second amplicon species is detectable in a second linearrange representing a third concentration of target nucleic acid in saidsample to a fourth concentration of target nucleic acid in said sample,and wherein: said first concentration < said third concentration < saidsecond concentration < said fourth concentration such that said firstand second linear ranges overlap and provide an extended dynamic rangefor determining the presence and amount of said target nucleic acid insaid sample, and wherein said detecting step is performed in a singlevessel.
 2. The method of claim 1, wherein a third differentiableamplicon species is generated.
 3. The method of claim 2, wherein saidthird amplicon is detectable in a linear range representing a fifthconcentration of target nucleic acid in said sample to a sixthconcentration of target nucleic acid in said sample.
 4. The method ofclaim 3, wherein: said first concentration < said third concentration <said second concentration < said fifth concentration < said fourthconcentration < said sixth concentrations such that said first, secondand third linear ranges overlap and provide an extended dynamic rangefor determining the presence and amount of said target nucleic acid insaid sample.
 5. The method of claim 1, wherein the at least twodifferentiable amplicon species are generated using a singleamplification oligomer that hybridizes to one strand of the targetnucleic acid and at least two amplification oligomers hybridize to acomplementary strand of the target nucleic acid, wherein said at leasttwo amplification oligomers hybridizing to a complementary strand ofsaid target nucleic acid are provided in amounts that differ by at leastone order of magnitude.
 6. The method of claim 5, wherein said at leasttwo amplification oligomers hybridizing to said complementary strand ofsaid target nucleic acid hybridize to distinct sequences of said targetnucleic acid, thereby generating from said target nucleic acid, adefined ratio of at least two differentiable amplicon species thatdiffer in length.
 7. The method of claim 5, wherein said at least twoamplification oligomers hybridizing to said complementary strand of saidtarget nucleic acid have distinct nucleic acid sequences, therebygenerating from said target nucleic acid, a defined ratio of at leasttwo differentiable amplicon species that differ in nucleic acidcomposition.
 8. The method of claim 7, wherein said at least twoamplification oligomers hybridizing to said complementary strand of saidtarget nucleic acid hybridize to the same sequence of said targetnucleic acid, wherein said at least two amplification oligomers havedistinct nucleic acid sequences selected from the group consisting of atleast one nucleotide mismatch, at least one nucleotide insertion, atleast one nucleotide deletion and combinations thereof, therebygenerating from said target nucleic acid, a defined ratio of at leasttwo differentiable amplicon species that differ in nucleic acidcomposition.
 9. The method of claim 1, wherein said two differentiableamplicon species are generated using an amplification method whereinsaid amplification method generates RNA amplicon species and DNAamplicon species to provide at least two differentiable amplicons thatdiffer in ribose sugar composition.
 10. The method of claim 9, whereinsaid amplification method is an isothermal amplification method thatuses at least one promoter-based amplification oligomer to generate agreater amount of RNA amplicon species that of DNA amplicon species. 11.The method of claim 1, wherein said two differentiable amplicon speciesare generated using a dNTP mixture wherein at least one of said dNTPspecies in said mix is provided in both a native form and in an analogform with one form in excess of the other form to provide at least twodifferentiable amplicons that differ in nucleic acid composition. 12.The method of claim 1, wherein said single vessel for performing step band said single vessel for performing step c are the same vessel. 13.The method of claim 1, wherein said amplification steps is anamplification method selected from the group consisting of: anisothermal amplification reaction, a cyclical amplification reaction, aTMA amplification reaction, a NASBA amplification reaction, a PCRamplification reaction, a Reverse Transcription (RT)-PCR amplificationreaction, a real time amplification reaction, a rolling circleamplification reaction, and a helicase dependent amplification reaction.